ABSTRACT
The oligoadenylate synthetase-ribonuclease L pathway is a major player in the interferon-induced antiviral defense mechanism of cells. Upon sensing viral dsRNA, 5'-phosphorylated 2',5'-oligoadenylates are synthesized, and subsequently activate latent RNase L. To determine the influence of 5'-phosphate end on the activation of human RNase L, four sets of 5'-phosphonate modified oligoadenylates were prepared on solid-phase. The ability of these 5'-modified oligoadenylates bearing shortened, isosteric and prolonged phosphonate linkages to activate RNase L was explored. We found that isosteric linkages and linkages prolonged by one atom were in general well tolerated by the enzyme with the EC50 values comparable to that of the natural activator. In contrast, linkages shortened by one atom or prolonged by two atoms exhibited decrease in the activity.
Subject(s)
Adenine Nucleotides/pharmacology , Endoribonucleases/metabolism , Oligoribonucleotides/pharmacology , Organophosphonates/pharmacology , Adenine Nucleotides/chemical synthesis , Adenine Nucleotides/chemistry , Dose-Response Relationship, Drug , Humans , Nucleic Acid Conformation , Oligoribonucleotides/chemical synthesis , Oligoribonucleotides/chemistry , Organophosphonates/chemical synthesis , Organophosphonates/chemistry , Structure-Activity RelationshipABSTRACT
The CRISPR system provides adaptive immunity against mobile genetic elements in prokaryotes. On binding invading RNA species, Type III CRISPR systems generate cyclic oligoadenylate (cOA) signalling molecules, potentiating a powerful immune response by activating downstream effector proteins, leading to viral clearance, cell dormancy or death. Here we describe the structure and mechanism of a cOA-activated CRISPR defence DNA endonuclease, CRISPR ancillary nuclease 1 (Can1). Can1 has a unique monomeric structure with two CRISPR associated Rossman fold (CARF) domains and two DNA nuclease-like domains. The crystal structure of the enzyme has been captured in the activated state, with a cyclic tetra-adenylate (cA4) molecule bound at the core of the protein. cA4 binding reorganises the structure to license a metal-dependent DNA nuclease activity specific for nicking of supercoiled DNA. DNA nicking by Can1 is predicted to slow down viral replication kinetics by leading to the collapse of DNA replication forks.
Subject(s)
Adenine Nucleotides/pharmacology , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Endonucleases/chemistry , Endonucleases/metabolism , Oligoribonucleotides/pharmacology , Binding Sites , DNA/metabolism , Models, Molecular , Plasmids/genetics , Protein Domains , Structural Homology, Protein , Thermus thermophilus/geneticsABSTRACT
Cytosolic nucleic acid-sensing pathways can be triggered to enhance immune response to cancer. In this study, we tested the antitumor activity of a unique RIG-I agonist, stem loop RNA (SLR) 14. In the immunogenic tumor models, we observed significant tumor growth delay and an extended survival in SLR14-treated mice. SLR14 also greatly improved antitumor efficacy of anti-PD1 antibody over single-agent treatment. SLR14 was mainly taken up by CD11b+ myeloid cells in the tumor microenvironment, and many genes associated with immune defense were significantly up-regulated after treatment, accompanied by increase in the number of CD8+ T lymphocytes, NK cells, and CD11b+ cells in SLR14-treated tumors. Strikingly, SLR14 dramatically inhibited nonimmunogenic B16 tumor growth, and the cured mice developed an immune memory. Furthermore, a systemic antitumor response was observed in both bilateral and tumor metastasis models. Collectively, our results demonstrate that SLR14 is a promising therapeutic RIG-I agonist for cancer treatment, either alone or in combination with existing immunotherapies.
Subject(s)
Antineoplastic Agents/pharmacology , Oligoribonucleotides/pharmacology , Receptors, Cell Surface/antagonists & inhibitors , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents, Immunological/pharmacology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Dose-Response Relationship, Drug , Gene Expression Profiling , Immunologic Memory , Injections, Intralesional , Male , Melanoma, Experimental , Mice , Oligoribonucleotides/administration & dosage , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Tumor Burden/drug effectsABSTRACT
Accumulating evidence has demonstrated that mitochondrial dysfunction is a prominent early event in the progression of Alzheimer's disease (AD). Whether protecting mitochondrial function can reduce amyloid-ß oligomer (AßO)-induced neurotoxicity in PS1V97L transgenic mice remains unknown. In this study, we examined the possible protective effects of honokiol (HKL) on mitochondrial dysfunction induced by AßOs in neurons, and cognitive function in AD PS1V97Ltransgenic mice. We determined that HKL increased mitochondrial sirtuin 3 (SIRT3) expression levels and activity, which in turn markedly improved ATP production and weakened mitochondrial reactive oxygen species production. We demonstrated that the enhanced energy metabolism and attenuated oxidative stress of HKL restores AßO-mediated mitochondrial dysfunction in vitro and in vivo. Consequently, memory deficits in the PS1V97L transgenic mice were rescued by HKL in the early stages. These results suggest that HKL has therapeutic potential for delaying the onset of AD symptoms by alleviating mitochondrial impairment and increasing hyperactivation of SIRT3 in the pathogenesis of preclinical AD.
Subject(s)
Alzheimer Disease/complications , Biphenyl Compounds/therapeutic use , Cognition Disorders/etiology , Enzyme Inhibitors/therapeutic use , Lignans/therapeutic use , Mitochondria/metabolism , Sirtuin 3/metabolism , Alzheimer Disease/genetics , Amyloid beta-Peptides/chemistry , Animals , Apoptosis/drug effects , Apoptosis/genetics , Cells, Cultured , Disease Models, Animal , Embryo, Mammalian , Female , Hippocampus/cytology , Male , Membrane Potential, Mitochondrial/drug effects , Membrane Potential, Mitochondrial/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mitochondria/drug effects , Neurons/drug effects , Neurons/ultrastructure , Oligoribonucleotides/pharmacology , Oxidative Stress/drug effects , Oxidative Stress/genetics , Presenilin-1/genetics , Rats , Rats, Sprague-DawleyABSTRACT
A novel supramolecular nanoparticle system with core-shell structure was designed based on ß-cyclodextrin-conjugated poly-l-lysine (PLCD) and hyaluronic acid for co-delivery of gene and chemotherapy agent targeting hepatocellular carcinoma (HCC). PLCD was synthesized by the conjugation of monoaldehyde activated ß-cyclodextrin with poly-l-lysine via Shiff's base reaction. Doxorubicin, as a model therapeutic drug, was included into the hydrophobic cavity of ß-cyclodextrin in PLCD through host-guest interaction. OligoRNA, as a model gene, was further condensed into the inclusion complexes by electrostatic interaction to form oligoRNA and doxorubicin co-loaded supramolecular nanoparticle system. Hyaluronic acid, which is often over-expressed by HCC cells, was coated on the surface of the above nanoparticles to construct HCC-targeted nanoparticle system. These nanoparticles had regular spherical shape with classic "core-shell" structure, and their size and zeta potential were 195.8nm and -22.7mV, respectively. The nanoparticles could effectively deliver doxorubicin and oligoRNA into HCC cells via CD44 receptor-mediated endocytosis and significantly inhibit the cell proliferation. In the nude mice bearing MHCC-97H tumor, the nanoparticles could be efficiently accumulated in the tumor, suggesting their strong hepatoma-targeting capability. These findings demonstrated that this novel supramolecular nanoparticle system had a promising potential for combining gene therapy and chemotherapy to treat HCC.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Hepatocellular/drug therapy , Drug Delivery Systems/methods , Gene Transfer Techniques , Liver Neoplasms/drug therapy , Nanoparticles/chemistry , Animals , Antineoplastic Agents/metabolism , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Doxorubicin/metabolism , Doxorubicin/pharmacology , Drug Compounding/methods , Drug Liberation , Drug Synergism , Gene Expression , Hep G2 Cells , Humans , Hyaluronan Receptors/genetics , Hyaluronan Receptors/metabolism , Hyaluronic Acid/chemistry , Kinetics , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Mice , Mice, Nude , Nanoparticles/ultrastructure , Oligoribonucleotides/metabolism , Oligoribonucleotides/pharmacology , Polylysine/chemistry , beta-Cyclodextrins/chemistryABSTRACT
BACKGROUND: Encapsidation, the process during which the genomic RNA of HIV is packaged into viral particles, is an attractive target for antiviral therapy. This study explores a novel nanotechnology-based strategy to inhibit HIV encapsidation by an RNA decoy mechanism. The design of the 16-mer oligoribonucleotide (RNA) decoy is based on the sequence of stem loop 3 (SL3) of the HIV packaging signal (Ψ). Recognition of the packaging signal is essential to the encapsidation process. It is theorized that the decoy RNA, by mimicking the packaging signal, will disrupt HIV packaging if efficiently delivered into lymphocytes by complexation with a carbosilane dendrimer. The aim of the study is to measure the uptake, toxicity, and antiviral activity of the dendrimer-RNA nanocomplex. MATERIALS AND METHODS: A dendriplex was formed between cationic carbosilane dendrimers and the RNA decoy. Uptake of the fluorescein-labeled RNA into MT4 lymphocytes was determined by flow cytometry and confocal microscopy. The cytoprotective effect (50% effective concentration [EC50]) and the effect on HIV replication were determined in vitro by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and viral load measurements, respectively. RESULTS: Flow cytometry and confocal imaging demonstrated efficient transfection of lymphocytes. The dendriplex containing the Ψ decoy showed some activity (EC50 =3.20 µM, selectivity index =8.4). However, there was no significant suppression of HIV viral load. CONCLUSION: Oligoribonucleotide decoys containing SL3 of the packaging sequence are efficiently delivered into lymphocytes by carbosilane dendrimers where they exhibit a modest cytoprotective effect against HIV infection.
Subject(s)
Anti-HIV Agents/pharmacology , Dendrimers/pharmacology , HIV-1/drug effects , Nanoparticles/chemistry , Oligoribonucleotides/pharmacology , Cell Death/drug effects , Cytoprotection/drug effects , Dendrimers/chemistry , Flow Cytometry , Humans , Inhibitory Concentration 50 , Nucleic Acid Conformation , RNA, Viral/chemistry , RNA, Viral/genetics , Silanes/chemistry , Transfection , Viral Load/drug effects , Virus Assembly/drug effectsABSTRACT
Induction therapy of proliferative lupus nephritis still requires the use of unselective immunosuppressive drugs with significant toxicities. In search of more specific drugs with equal efficacy but fewer side effects we considered blocking pro-inflammatory chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) and homeostatic chemokine stromal cell-derived factor-1 (SDF-1/CXCL12), which both contribute to the onset and progression of proliferative lupus nephritis yet through different mechanisms. We hypothesized that dual antagonism could be as potent on lupus nephritis as the unselective immunosuppressant cyclophosphamide (CYC). We estimated serum levels of CCL2 and CXCL12 in patients with SLE (n=99) and compared the results with healthy individuals (n=21). In order to prove our hypothesis we used l-enantiomeric RNA Spiegelmer® chemokine antagonists, i.e. the CCL2-specific mNOX-E36 and the CXCL12-specific NOX-A12 to treat female MRL/lpr mice from week 12 to 20 of age with either anti-CXCL12 or anti-CCL2 alone or both. SLE patients showed elevated serum levels of CCL2 but not of CXCL12. Female MRL/lpr mice treated with dual blockade showed significantly more effective than either monotherapy in preventing proteinuria, immune complex glomerulonephritis, and renal excretory failure and the results are at par with CYC treatment. Dual blockade reduced leukocyte counts and renal IL-6, IL-12p40, CCL-5, CCL-2 and CCR-2 mRNA expression. Dual blockade of CCL2 and CXCL12 can be as potent as CYC to suppress the progression of proliferative lupus nephritis probably because the respective chemokine targets mediate different disease pathomechanisms, i.e. systemic autoimmunity and peripheral tissue inflammation.
Subject(s)
Chemokine CCL2/antagonists & inhibitors , Chemokine CXCL12/antagonists & inhibitors , Cyclophosphamide/pharmacology , Lupus Nephritis/drug therapy , Oligoribonucleotides/pharmacology , Adult , Animals , Chemokine CCL2/genetics , Chemokine CCL2/metabolism , Chemokine CXCL12/genetics , Chemokine CXCL12/metabolism , Enzyme-Linked Immunosorbent Assay , Female , Gene Expression/genetics , Glomerulonephritis/prevention & control , Homeostasis/genetics , Humans , Immunosuppressive Agents/pharmacology , Kidney/drug effects , Kidney/metabolism , Kidney/pathology , Lupus Erythematosus, Systemic/blood , Lupus Nephritis/genetics , Lupus Nephritis/metabolism , Male , Mice, Inbred MRL lpr , Middle Aged , Oligoribonucleotides/genetics , Oligoribonucleotides/metabolism , Proteinuria/prevention & control , Reverse Transcriptase Polymerase Chain Reaction , Treatment OutcomeABSTRACT
Differences in sensitivity of monkeys and humans to antisense oligonucleotide (ASO)-induced complement alternative pathway (AP) activation were evaluated in monkeys, humans, and in serum using biochemical assays. Transient AP activation was evident in monkeys at higher doses of two 2'-O-methoxyethyl (2'-MOE) ASOs (ISIS 426115 and ISIS 183750). No evidence of AP activation was observed in humans for either ASO, even with plasma ASO concentrations that reached the threshold for activation in monkeys. The absence of complement activation in humans is consistent with a query of the Isis Clinical Safety Database containing 767 subjects. The in vivo difference in sensitivity was confirmed in vitro, as monkey and human serum exposed to increasing concentrations of ASO indicated that monkeys were more sensitive to AP activation with this class of compounds. The mechanistic basis for the greater sensitivity of monkeys to AP activation by 2'-MOE ASO was evaluated using purified human or monkey factor H protein. The binding affinities between a representative 2'-MOE ASO and either purified protein are similar. However, the IC50 of fluid-phase complement inhibition for monkey factor H is about 3-fold greater than that for human protein using either monkey serum or factor H-depleted human serum. Interestingly, there is a sequence variant in the monkey complement factor H gene similar to a single nucleotide polymorphism in humans that is correlated with decreased factor H protein function. These findings show that monkeys are more sensitive to 2'-MOE ASO-mediated complement activation than humans likely because of differences in factor H inhibitory capacity.
Subject(s)
Complement Activation/drug effects , Complement Activation/physiology , Complement Factor H/genetics , Comprehension , Oligonucleotides, Antisense/pharmacology , Oligoribonucleotides/pharmacology , Adolescent , Adult , Amino Acid Sequence , Animals , Dose-Response Relationship, Drug , Double-Blind Method , Female , Humans , Macaca fascicularis , Male , Middle Aged , Molecular Sequence Data , Oligonucleotides , Oligonucleotides, Antisense/genetics , Oligoribonucleotides/genetics , Young AdultABSTRACT
Recently it was reported that microRNA from the miR-17 ~ 92 family may have a key role in chronic lymphocytic leukemia (CLL). Here, we designed specific oligonucleotides to target endogenous miR-17 (antagomiR17). In-vitro administration of antagomiR17 effectively reduced miR-17 expression and the proliferation of CLL-like MEC-1 cells. When injected in-vivo in tumor generated by the MEC-1 cells in SCID mice, antagomiR17 dramatically reduced tumor growth and significantly increase survival. Altogether, our results provide the rationale for the use of antagomiR17 as a novel potential therapeutic tool in CLL and in other lymphoproliferative disorders where miR-17 has a driver role in tumor progression.
Subject(s)
Antineoplastic Agents/pharmacology , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , MicroRNAs/antagonists & inhibitors , Oligoribonucleotides/pharmacology , Animals , Cell Line, Tumor , Humans , Mice , Mice, SCID , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
The ability of innate immune cells to sense and respond to impending danger varies by anatomical location. The liver is considered tolerogenic but is still capable of mounting a successful immune response to clear various infections. To understand whether hepatic immune cells tune their response to different infectious challenges, we probed mononuclear cells purified from human healthy and diseased livers with distinct pathogen-associated molecules. We discovered that only the TLR8 agonist ssRNA40 selectively activated liver-resident innate immune cells to produce substantial quantities of IFN-γ. We identified CD161(Bright) mucosal-associated invariant T (MAIT) and CD56(Bright) NK cells as the responding liver-resident innate immune cells. Their activation was not directly induced by the TLR8 agonist but was dependent on IL-12 and IL-18 production by ssRNA40-activated intrahepatic monocytes. Importantly, the ssRNA40-induced cytokine-dependent activation of MAIT cells mirrored responses induced by bacteria, i.e., generating a selective production of high levels of IFN-γ, without the concomitant production of TNF-α or IL-17A. The intrahepatic IFN-γ production could be detected not only in healthy livers, but also in HBV- or HCV-infected livers. In conclusion, the human liver harbors a network of immune cells able to modulate their immunological responses to different pathogen-associated molecules. Their ability to generate a strong production of IFN-γ upon stimulation with TLR8 agonist opens new therapeutic opportunities for the treatment of diverse liver pathologies.
Subject(s)
Adjuvants, Immunologic/pharmacology , Immunity, Innate/drug effects , Leukocytes, Mononuclear/drug effects , Liver/drug effects , Oligoribonucleotides/pharmacology , Toll-Like Receptor 8/agonists , Up-Regulation/drug effects , Cells, Cultured , Coculture Techniques , Enterococcus faecalis/immunology , Enterococcus faecalis/metabolism , Enterococcus faecalis/pathogenicity , Escherichia coli/immunology , Escherichia coli/metabolism , Escherichia coli/pathogenicity , Hepacivirus/immunology , Hepacivirus/pathogenicity , Hepatitis B/immunology , Hepatitis B/metabolism , Hepatitis B/pathology , Hepatitis B/virology , Hepatitis B virus/immunology , Hepatitis B virus/pathogenicity , Hepatitis C/immunology , Hepatitis C/metabolism , Hepatitis C/pathology , Hepatitis C/virology , Humans , Interferon-gamma Release Tests , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/pathology , Liver/immunology , Liver/microbiology , Liver/pathology , Monocytes/drug effects , Monocytes/immunology , Monocytes/metabolism , Pseudomonas aeruginosa/immunology , Pseudomonas aeruginosa/metabolism , Pseudomonas aeruginosa/pathogenicity , Riboflavin/biosynthesis , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Toll-Like Receptor 8/metabolismABSTRACT
TLRs 7 and 8 are pattern recognition receptors controlling antiviral host defense or autoimmune diseases. Apart from foreign and host RNA, synthetic RNA oligoribonucleotides (ORN) or small molecules of the imidazoquinoline family activate TLR7 and 8 and are being developed as therapeutic agonists. The structure-function relationships for RNA ORN and imidazoquinoline sensing and consequent downstream signaling by human TLR7 and TLR8 are unknown. Proteome- and genome-wide analyses in primary human monocyte-derived dendritic cells here showed that TLR8 sensing of RNA ORN versus imidazoquinoline translates to ligand-specific differential phosphorylation and transcriptional events. In addition, TLR7 and 8 ectodomains were found to discriminate between RNA ORN and imidazoquinolines by overlapping and nonoverlapping recognition sites to which murine loss-of-function mutations and human naturally occurring hyporesponsive polymorphisms map. Our data suggest TLR7 and TLR8 can signal in two different "modes" depending on the class of ligand. Considering RNA ORN and imidazoquinolines have been regarded as functionally interchangeable, our study highlights important functional incongruities whose understanding will be important for developing TLR7 or 8 therapeutics with desirable effector and safety profiles for in vivo application.
Subject(s)
Oligoribonucleotides/pharmacology , Quinolinium Compounds/pharmacology , RNA/pharmacology , Signal Transduction/drug effects , Toll-Like Receptor 7/immunology , Toll-Like Receptor 8/immunology , Animals , HEK293 Cells , Humans , Mice , Oligoribonucleotides/chemistry , Protein Structure, Tertiary , Quinolinium Compounds/chemistry , RNA/chemistry , Signal Transduction/genetics , Signal Transduction/immunology , Toll-Like Receptor 7/genetics , Toll-Like Receptor 8/geneticsABSTRACT
Endoplasmic reticulum (ER) stress has been implicated in a number of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). MicroRNAs are small ribonucleic acids which can modulate protein expression by binding to the 3'UTR of target mRNAs. We recently identified increased miR-29a expression in response to ER stress in neurons, with members of the miR-29 family implicated in cancer and neurodegeneration. We found high expression of miR-29a in the mouse brain and spinal cord by quantitative PCR analysis and increased expression of miR-29a in the spinal cord of SOD1(G93A) transgenic mice, a mouse model of familial ALS. In situ hybridisation experiments revealed increased miR-29a expression in the lumbar spinal cord of SOD1(G93A) transgenic mice from postnatal day 70 onward when compared to wild-type mice. miR-29a knockdown was achieved in the CNS in vivo after a single intracerebroventricular injection of a miR-29a-specific antagomir. While analysis of disease progression and motor function could not identify a significant alteration in ALS disease manifestations, a trend towards increased lifespan was observed in male SOD1(G93A) mice. These findings demonstrate that miR-29a may act as a marker for disease progression in SOD1(G93A) mice, and provide first proof-of-concept for a therapeutic modulation of miR-29a function in ALS.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , MicroRNAs/metabolism , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Animals , Brain/drug effects , Brain/metabolism , Male , Mice , Mice, Inbred C57BL , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , Oligoribonucleotides/pharmacology , Spinal Cord/drug effects , Spinal Cord/metabolism , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Superoxide Dismutase-1ABSTRACT
RNase L is a cellular endoribonuclease that is activated by 2',5'-linked oligoadenylates (2-5A), which are unique and specific ligands synthesized by a family of interferon-inducible, dsRNA-activated enzymes named oligoadenylate synthetases. In the typical antiviral pathway, activated RNase L degrades viral and cellular RNAs, thus limiting viral replication and spread. Although the antiviral activity of RNase L has been demonstrated for several RNA viruses, there is little evidence regarding its role against DNA viruses. In the present study, the potential antiviral activity of RNase L against hepatitis B virus (HBV) was explored utilizing the recently reported infection protocol based on human hepatoma HepG2 cells stably complemented with the virus entry factor NTCP. Viral replication and expression in this cell type was markedly inhibited by poly(I:C)- or 2-5A-mediated activation of RNase L; however, the inhibition was significantly reversed by RNase L knockdown. Further analysis in HBV1.2-transfected Huh-7 hepatoma cells indicated that the antiviral activity of RNase L depends on its ribonuclease function. We also provide evidence for the specific roles of OAS family members in this process. These results suggest that HBV replication can be regulated through interferon-mediated RNA decay pathways and that activation of these host antiviral factors may represent a novel therapeutic strategy for HBV infection.
Subject(s)
Endoribonucleases/metabolism , Hepatitis B virus/physiology , Virus Replication , Adenine Nucleotides/pharmacology , Cell Line , Endoribonucleases/genetics , Enzyme Activation/drug effects , Gene Expression , Gene Knockdown Techniques , Hepatitis B virus/drug effects , Humans , Isoenzymes , Ligands , Oligoribonucleotides/pharmacology , Poly I-C/pharmacology , Virus Replication/drug effectsABSTRACT
OBJECTIVE: Vascular precursor cells with angiogenic potentials are important for tissue repair, which is impaired in diabetes mellitus. MicroRNAs are recently discovered key regulators of gene expression, but their role in vascular precursor cell-mediated angiogenesis in diabetes mellitus is unknown. We tested the hypothesis that the microRNA miR-27b rescues impaired bone marrow-derived angiogenic cell (BMAC) function in vitro and in vivo in type 2 diabetic mice. APPROACH AND RESULTS: BMACs from adult male type 2 diabetic db/db and from normal littermate db/+ mice were used. miR-27b expression was decreased in db/db BMACs. miR-27b mimic improved db/db BMAC function, including proliferation, adhesion, tube formation, and delayed apoptosis, but it did not affect migration. Elevated thrombospondin-1 (TSP-1) protein in db/db BMACs was suppressed on miR-27b mimic transfection. Inhibition of miR-27b in db/+ BMACs reduced angiogenesis, which was reversed by TSP-1 small interfering RNA (siRNA). miR-27b suppressed the pro-oxidant protein p66(shc) and mitochondrial oxidative stress, contributing to its protection of BMAC function. miR-27b also suppressed semaphorin 6A to improve BMAC function in diabetes mellitus. Luciferase binding assay suggested that miR-27b directly targeted TSP-1, TSP-2, p66(shc), and semaphorin 6A. miR-27b improved topical cell therapy of diabetic BMACs on diabetic skin wound closure, with a concomitant augmentation of wound perfusion and capillary formation. Normal BMAC therapy with miR-27b inhibition demonstrated reduced efficacy in wound closure, perfusion, and capillary formation. Local miR-27b delivery partly improved wound healing in diabetic mice. CONCLUSIONS: miR-27b rescues impaired BMAC angiogenesis via TSP-1 suppression, semaphorin 6A expression, and p66shc-dependent mitochondrial oxidative stress and improves BMAC therapy in wound healing in type 2 diabetic mice.
Subject(s)
Bone Marrow Cells/metabolism , Diabetes Mellitus, Type 2/metabolism , MicroRNAs/metabolism , Neovascularization, Physiologic , Stem Cells/metabolism , Wound Healing , Animals , Bone Marrow Cells/pathology , CD36 Antigens/deficiency , CD36 Antigens/genetics , CD47 Antigen/genetics , CD47 Antigen/metabolism , Case-Control Studies , Cells, Cultured , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/pathology , Diabetes Mellitus, Type 2/physiopathology , Diabetes Mellitus, Type 2/therapy , Disease Models, Animal , Female , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , MicroRNAs/antagonists & inhibitors , MicroRNAs/blood , MicroRNAs/genetics , Middle Aged , Mitochondria/metabolism , Oligoribonucleotides/pharmacology , Oxidative Stress , RNA Interference , Semaphorins/genetics , Semaphorins/metabolism , Shc Signaling Adaptor Proteins/genetics , Shc Signaling Adaptor Proteins/metabolism , Signal Transduction , Src Homology 2 Domain-Containing, Transforming Protein 1 , Stem Cells/pathology , Thrombospondin 1/genetics , Thrombospondin 1/metabolism , Thrombospondins/genetics , Thrombospondins/metabolism , Time Factors , TransfectionABSTRACT
AIMS: Dysregulation of the bone morphogenetic protein receptor type 2 (BMPR2) is a hallmark feature that has been described in several forms of pulmonary hypertension. We recently identified the microRNA miR-20a within a highly conserved pathway as a regulator of the expression of BMPR2. To address the pathophysiological relevance of this pathway in vivo, we employed antagomiR-20a and investigated whether specific inhibition of miR-20a could restore functional levels of BMPR2 and, in turn, might prevent pulmonary arterial vascular remodelling. METHODS AND RESULTS: For specific inhibition of miR-20a, cholesterol-modified RNA oligonucleotides (antagomiR-20a) were synthesized. The experiments in mice were performed by using the hypoxia-induced mouse model for pulmonary hypertension and animal tissues were analysed for right ventricular hypertrophy and pulmonary arterial vascular remodelling. Treatment with antagomiR-20a enhanced the expression levels of BMPR2 in lung tissues; moreover, antagomiR-20a significantly reduced wall thickness and luminal occlusion of small pulmonary arteries and reduced right ventricular hypertrophy. To assess BMPR2 signalling and proliferation, we performed in vitro experiments with human pulmonary arterial smooth muscle cells (HPASMCs). Transfection of HPASMCs with antagomiR-20a resulted in activation of downstream targets of BMPR2 showing increased activation of Id-1 and Id-2. Proliferation of HPASMCs was found to be reduced upon transfection with antagomiR-20a. CONCLUSION: This is the first report showing that miR-20a can be specifically targeted in an in vivo model for pulmonary hypertension. Our data emphasize that treatment with antagomiR-20a restores functional levels of BMPR2 in pulmonary arteries and prevents the development of vascular remodelling.
Subject(s)
Antihypertensive Agents/pharmacology , Bone Morphogenetic Protein Receptors, Type II/physiology , Hypertension, Pulmonary/prevention & control , Hypoxia/complications , MicroRNAs/antagonists & inhibitors , Vascular Remodeling/drug effects , Animals , Bone Morphogenetic Protein Receptors, Type II/metabolism , Cell Proliferation/physiology , Cholesterol/analogs & derivatives , Cholesterol/pharmacology , Coronary Circulation/physiology , Disease Models, Animal , Hypertension, Pulmonary/etiology , Hypertrophy, Right Ventricular/physiopathology , In Vitro Techniques , Male , Mice , Muscle, Smooth, Vascular/cytology , Oligoribonucleotides/pharmacology , Pulmonary Circulation/physiology , Signal Transduction/physiology , TransfectionABSTRACT
5'-Triphosphate 2'-5'-oligoadenylate (2-5A) is the central player in the 2-5A system that is an innate immunity pathway in response to the presence of infectious agents. Intracellular endoribonuclease RNase L activated by 2-5A cleaves viral and cellular RNA resulting in apoptosis. The major limitations of 2-5A for therapeutic applications is the short biological half-life and poor cellular uptake. Modification of 2-5A with biolabile and lipophilic groups that facilitate its uptake, increase its in vivo stability and release the parent 2-5A drug in an intact form offer an alternative approach to therapeutic use of 2-5A. Here we have synthesized the trimeric and tetrameric 2-5A species bearing hydrophobic and enzymolabile pivaloyloxymethyl groups at 3'-positions and a triphosphate at the 5'-end. Both analogs were able to activate RNase L and the production of the trimer 2-5A (the most active) was scaled up to the milligram scale for antiviral evaluation in cells infected by influenza virus or respiratory syncytial virus. The trimer analog demonstrated some significant antiviral activity.
Subject(s)
Adenine Nucleotides/chemistry , Antiviral Agents/chemical synthesis , Endoribonucleases/chemistry , Oligoribonucleotides/chemistry , Adenine Nucleotides/chemical synthesis , Adenine Nucleotides/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Cell Line, Tumor , Endoribonucleases/metabolism , Fluorescence Resonance Energy Transfer , Humans , Influenza A virus/drug effects , Oligoribonucleotides/chemical synthesis , Oligoribonucleotides/pharmacology , Polymerization , Solid-Phase Synthesis TechniquesABSTRACT
Serious complications as a result of type 2 diabetes mellitus (T2DM) are becoming a major health concern. In the present study, it was hypothesized that telomerase activity is upregulated in vascular smooth muscle cells (VSMCs) during proliferation in T2DM and that the application of telomerase inhibitors impedes the proliferation of VSMCs in vitro. Male Wistar rats were randomly allocated into the normal control (NC) or diabetic (DM) group. Diabetes was induced by highfat feeding and a low dose of streptozotocin (STZ; 30 mg/kg). Primary VSMC cultures were exposed to normal (5.5 mM) or high (25 mM) glucose and insulin (100 nM) in the presence and absence of various concentrations of antisense oligoribonucleotides (ASODNs) for varying lengths of time. Telomerase activity and the proliferation of VSMCs were measured. Results showed that there was a significant increase in the levels of fasting glucose, insulin, triglycerides (TG) and free fatty acids (FFAs) in the diabetic group. Telomerase activity and the proliferation of VSMCs were significantly higher in the diabetic group in vivo and in the high glucose and insulin (HGI)-treated group in vitro (P<0.01). ASODNs significantly inhibited the proliferation of VSMCs in a concentration- and timedependent manner (P<0.01). In conclusion, hyperglycemia and hyperinsulinemia stimulate telomerase activity and the proliferation of VSMCs, while the inhibition of telomerase activity reduces the proliferation of VSMCs, indicating that telomerase may be involved in the pathological process of diabetic vascular disease.
Subject(s)
Diabetes Mellitus, Experimental/enzymology , Diabetes Mellitus, Type 2/enzymology , Diabetes Mellitus, Type 2/pathology , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/enzymology , Myocytes, Smooth Muscle/pathology , Telomerase/metabolism , Animals , Blood Glucose/metabolism , Cell Proliferation , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 2/blood , Fatty Acids/blood , Male , Oligoribonucleotides/pharmacology , Rats , Rats, Wistar , Triglycerides/bloodABSTRACT
Oligonucleotides containing an immune-stimulatory motif and an immune-regulatory motif act as antagonists of Toll-like receptor (TLR)7 and TLR9. In the present study, we designed and synthesized oligonucleotide-based antagonists of TLR7, 8 and 9 containing a 7-deaza-dG or arabino-G modification in the immune-stimulatory motif and 2'-O-methylribonucleotides as the immune-regulatory motif. We evaluated the biological properties of these novel synthetic oligoribonucleotides as antagonists of TLRs 7, 8 and 9 in murine and human cell-based assays and in vivo in mice and non-human primates. In HEK293, mouse and human cell-based assays, the antagonist compounds inhibited signaling pathways and production of a broad range of cytokines, including tumour necrosis factor alpha (TNF-α), interleukin (IL)-12, IL-6, interferon (IFN)-α, IL-1ß and interferon gamma-induced protein (IP)-10, mediated by TLR7, 8 and 9. In vivo in mice, the antagonist compounds inhibited TLR7- and TLR9-mediated cytokine induction in a dose- and time-dependent fashion. Peripheral blood mononuclear cells (PBMCs) obtained from antagonist compound-treated monkeys secreted lower levels of TLR7-, 8- and 9-mediated cytokines than did PBMCs taken before antagonist administration. The antagonist compounds described herein provide novel agents for the potential treatment of autoimmune and inflammatory diseases.
Subject(s)
Toll-Like Receptor 7/antagonists & inhibitors , Toll-Like Receptor 8/antagonists & inhibitors , Toll-Like Receptor 9/antagonists & inhibitors , Animals , Cells, Cultured , Cytokines/biosynthesis , Female , HEK293 Cells , Humans , Lupus Erythematosus, Systemic/immunology , Macaca fascicularis , Male , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , Oligoribonucleotides/chemistry , Oligoribonucleotides/pharmacology , Signal Transduction/drug effects , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 8/metabolism , Toll-Like Receptor 9/metabolism , Toll-Like Receptors/agonists , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Anemia of chronic inflammation is the most prevalent form of anemia in hospitalized patients. A hallmark of this disease is the intracellular sequestration of iron. This is a consequence of hepcidin-induced internalization and subsequent degradation of ferroportin, the hepcidin receptor and only known iron-export protein. This study describes the characterization of novel anti-hepcidin compound NOX-H94, a structured L-oligoribonucleotide that binds human hepcidin with high affinity (Kd = 0.65 ± 0.06 nmol/L). In J774A.1 macrophages, NOX-H94 blocked hepcidin-induced ferroportin degradation and ferritin expression (half maximal inhibitory concentration = 19.8 ± 4.6 nmol/L). In an acute cynomolgus monkey model of interleukin 6 (IL-6)-induced hypoferremia, NOX-H94 inhibited serum iron reduction completely. In a subchronic model of IL-6-induced anemia, NOX-H94 inhibited the decrease in hemoglobin concentration. We conclude that NOX-H94 protects ferroportin from hepcidin-induced degradation. Therefore, this pharmacologic approach may represent an interesting treatment option for patients suffering from anemia of chronic inflammation.
Subject(s)
Anemia/drug therapy , Anemia/etiology , Inflammation/complications , Inflammation/drug therapy , Oligoribonucleotides/therapeutic use , Anemia/pathology , Animals , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Antimicrobial Cationic Peptides/antagonists & inhibitors , Cells, Cultured , Disease Models, Animal , Drug Evaluation, Preclinical , Erythrocytes/drug effects , Erythrocytes/metabolism , Hemoglobins/analysis , Hemoglobins/drug effects , Hepcidins , Interleukin-6/administration & dosage , Interleukin-6/adverse effects , Iron/blood , Iron/metabolism , Iron Metabolism Disorders/chemically induced , Macaca fascicularis , Macrophages/drug effects , Macrophages/metabolism , Male , Mice , Oligoribonucleotides/administration & dosage , Oligoribonucleotides/pharmacologyABSTRACT
microRNAs (miRNAs) are small noncoding RNAs that have been linked to a number of disease-related signal transduction pathways. Several studies indicate that they are also involved in nociception. It is not clear, however, which miRNAs are important and which genes are modulated by miRNA-associated mechanisms. This study focuses on the regulation and function of the central nervous system (CNS)-specific miRNA-124a in the spinal cord of mice in a formalin model of inflammatory nociception. miRNA-124a is constitutively expressed in the spinal cord of mice, particularly in neurons of the dorsal horn. Peripheral noxious stimulation with formalin led to significant down-regulation of its expression. Knock-down of miRNA-124a by intravenous administration of a specific miRNA-124a inhibitor further increased the nociceptive behavior associated with an upregulation of the pain-relevant miRNA-124a target MeCP2 and proinflammatory marker genes. In contrast, administration of a miRNA-124a mimic counteracted these effects and decreased nociception by down-regulation of the target gene. In conclusion, our results indicate that miRNA-124a is involved in inflammatory nociception by regulation of relevant target proteins and might therefore constitute a novel target for anti-inflammatory therapy.