Stimulation of mouse hematopoietic stem cells by angiogenin and DNA preparations.

Immature hematopoietic progenitors are a constant source for renewal of hemocyte populations and the basic component of the tissue and cell repair apparatus. A unique property of these cells of internalizing extracellular double-stranded DNA has been previously shown. The leukostimulatory effect demonstrated in our pioneering studies was considered to be due to the feature of this cell. In the present research, we have analyzed the effects of DNA genome reconstructor preparation (DNAgr), DNAmix, and human recombinant angiogenin on both hematopoietic stem cells and multipotent progenitors. Treatment with bone marrow cells of experimental mice with these preparations stimulates colony formation by hematopoietic stem cells and proliferation of multipotent descendants. The main lineage responsible for this is the granulocyte-macrophage hematopoietic lineage. Using fluorescent microscopy as well as FACS assay, co-localization of primitive c-Kit- and Sca-1-positive progenitors and the TAMRA-labeled double-stranded DNA has been shown. Human recombinant angiogenin was used as a reference agent. Cells with specific markers were quantified in intact bone marrow and colonies grown in the presence of inducers. Quantitative analysis revealed that a total of 14,000 fragment copies of 500 bp, which is 0.2% of the haploid genome, can be delivered into early progenitors. Extracellular double-stranded DNA fragments stimulated the colony formation in early hematopoietic progenitors from the bone marrow, which assumed their effect on cells in G0. The observed number of Sca1+/c-Kit+ cells in colonies testifies to the possibility of both symmetrical and asymmetrical division of the initial hematopoietic stem cell and its progeny.

Enhancement of the activity of the antimicrobial peptides HNP1 and LL-37 by bovine pancreatic ribonuclease A.

Background: HNP1, LL-37, and HBD1 are antimicrobial against Escherichia coli ATCC 25922 at the standard inoculum but less active at higher inocula.   Methods: The virtual colony count (VCC) microbiological assay was adapted for high inocula and the addition of yeast tRNA and bovine pancreatic ribonuclease A (RNase).  96-well plates were read for 12 hours in a Tecan Infinite M1000 plate reader and photographed under 10x magnification.    Results: Adding tRNA 1:1 wt/wt to HNP1 at the standard inoculum almost completely abrogated activity.  Adding RNase 1:1 to HNP1 at the standard inoculum of 5x10 5 CFU/mL did not enhance activity.  Increasing the inoculum to 6.25x10 7 CFU/mL almost abrogated HNP1 activity.  However, adding RNase 25:1 to HNP1 enhanced activity at the highest tested concentration of HNP1.  Adding both tRNA and RNase resulted in enhanced activity, indicating that the enhancement effect of RNase overwhelms the inhibiting effect of tRNA when both are present.  HBD1 activity at the standard inoculum was almost completely abrogated by the addition of tRNA, but LL-37 activity was only slightly inhibited by tRNA.  At the high inoculum, LL-37 activity was enhanced by RNase.  HBD1 activity was not enhanced by RNase.  RNase was not antimicrobial in the absence of antimicrobial peptides.  Cell clumps were observed at the high inoculum in the presence of all three antimicrobial peptides and at the standard inoculum in the presence of HNP1+tRNA and HBD1+tRNA.    Conclusions: Antimicrobial peptide-ribonuclease combinations have the potential to be active against high cell concentrations, conditions where the antimicrobial agent alone is relatively ineffective.

Osteoclasts protect bone blood vessels against senescence through the angiogenin/plexin-B2 axis.

Synthetic glucocorticoids (GCs), one of the most effective treatments for chronic inflammatory and autoimmune conditions in children, have adverse effects on the growing skeleton. GCs inhibit angiogenesis in growing bone, but the underlying mechanisms remain unclear. Here, we show that GC treatment in young mice induces vascular endothelial cell senescence in metaphysis of long bone, and that inhibition of endothelial cell senescence improves GC-impaired bone angiogenesis with coupled osteogenesis. We identify angiogenin (ANG), a ribonuclease with pro-angiogenic activity, secreted by osteoclasts as a key factor for protecting the neighboring vascular cells against senescence. ANG maintains the proliferative activity of endothelial cells through plexin-B2 (PLXNB2)-mediated transcription of ribosomal RNA (rRNA). GC treatment inhibits ANG production by suppressing osteoclast formation in metaphysis, resulting in impaired endothelial cell rRNA transcription and subsequent cellular senescence. These findings reveal the role of metaphyseal blood vessel senescence in mediating the action of GCs on growing skeleton and establish the ANG/PLXNB2 axis as a molecular basis for the osteoclast-vascular interplay in skeletal angiogenesis.

Angiogenin maintains gut microbe homeostasis by balancing α-Proteobacteria and Lachnospiraceae.

OBJECTIVE: Antimicrobial peptides (AMPs) play essential roles in maintaining gut health and are associated with IBD. This study is to elucidate the effect of angiogenin (ANG), an intestine-secreted AMP, on gut microbiota and its relevance with IBD. DESIGN: The effect of ANG on microbiota and its contribution to colitis were evaluated in different colitis models with co-housing and faecal microbiota transplantation. ANG-regulated bacteria were determined by 16S rDNA sequencing and their functions in colitis were analysed by bacterial colonisation. The species-specific antimicrobial activity of ANG and its underlying mechanism were further investigated with microbiological and biochemical methods. ANG level and the key bacteria were characterised in IBD faecal samples. RESULTS: ANG regulated microbiota composition and inhibited intestinal inflammation. Specifically, Ang1 deficiency in mice led to a decrease in the protective gut commensal strains of Lachnospiraceae but an increase in the colitogenic strains of α-Proteobacteria. Direct binding of ANG to α-Proteobacteria resulted in lethal disruption of bacterial membrane integrity, and consequently promoted the growth of Lachnospiraceae, which otherwise was antagonised by α-Proteobacteria. Oral administration of ANG1 reversed the dysbiosis and attenuated the severity of colitis in Ang1-deficient mice. The correlation among ANG, the identified bacteria and IBD status was established in patients. CONCLUSION: These findings demonstrate a novel role of ANG in shaping gut microbe composition and thus maintaining gut health, suggesting that the ANG-microbiota axis could be developed as a potential preventive and/or therapeutic approach for dysbiosis-related gut diseases.

Hierarchical Self-assembly of Discrete Metal-Organic Cages into Supramolecular Nanoparticles for Intracellular Protein Delivery.

Hierarchical self-assembly (HAS) is a powerful approach to create supramolecular nanostructures for biomedical applications. This potency, however, is generally challenged by the difficulty of controlling the HAS of biomacromolecules and the functionality of resulted HAS nanostructures. Herein, we report a modular approach for controlling the HAS of discrete metal-organic cages (MOC) into supramolecular nanoparticles, and its potential for intracellular protein delivery and cell-fate specification. The hierarchical coordination-driven self-assembly of adamantane-functionalized M12 L24 MOC (Ada-MOC) and the host-guest interaction of Ada-MOC with ß-cyclodextrin-conjugated polyethylenimine (PEI-ßCD) afford supramolecular nanoparticles in a controllable manner. HAS maintains high efficiency and orthogonality in the presence of protein, enabling the encapsulation of protein into the nanoparticles for intracellular protein delivery for therapeutic application and CRISPR/Cas9 genome editing.

Targeted Delivery of DNA Framework-Encapsulated Native Therapeutic Protein into Cancer Cells.

A protein-based therapy is significantly challenged by the successful delivery of native proteins into the targeted cancer cells. We address this challenge here using an all-sealed divalent aptamer tetrahedral DNA framework (asdTDF) delivery platform, in which the protein drug is encapsulated inside the cavity of the framework stoichiometrically via a reversible chemical bond. The ligase-assisted seal of the nicks results in highly enhanced TDF stability of the against nuclease digestion to effectively protect the therapeutic protein from degradation. In addition, the divalent aptamer sequences incorporated into the framework favor it with a target-specific and efficient delivery capability. Importantly, upon being readily delivered into the targeted cancer cells, endogenous glutathione can trigger the release of the native therapeutic protein from the TDF in a traceless fashion by cleaving the reversible chemical bond, thereby leading to effective apoptosis of the specific cancer cells.

Characterization of Naturally Occurring Bioactive Factor Mixtures for Bone Regeneration.

In this study, the bone-regenerative potential of bioactive factors derived from adipose tissue, platelet-rich plasma (PRP) and conditioned medium from hypoxia-treated human telomerase immortalized bone-marrow-derived mesenchymal stem cells (hTERT-MSC) was investigated in vitro with the aim to develop cost-effective and efficient bone substitutes for optimized regeneration of bone defects. Adipose tissue was harvested from human donors undergoing reconstructive surgery, and adipose tissue extract (ATE) was prepared. Platelet lysates (PL) were produced by repeated freeze-thaw cycles of PRP, and hypoxia-conditioned medium (HCM) was obtained by culturing human telomerase immortalized bone-marrow-derived mesenchymal stromal cells for 5 days with 1% O2. Besides analysis by cytokine and angiogenesis arrays, ELISA was performed. Angiogenic potential was investigated in cocultures of bone-marrow-derived (BM)-MSC and human umbilical vein endothelial cells. Multiple angiogenic proteins and cytokines were detected in all growth factor mixtures. HCM and ATE contained high amounts of angiogenin and CCL2/MCP-1, whereas PL contained high amounts of IGFBP-1. Culturing cells with HCM and ATE significantly increased specific ALP activity of BM-MSC as well as tubule length and junctions of endothelial networks, indicating osteogenic and angiogenic stimulation. To achieve a synergism between chemoattractive potential and osteogenic and angiogenic differentiation capacity, a combination of different growth factors appears promising for potential clinical applications.

Chemosensitization of prostate cancer stem cells in mice by angiogenin and plexin-B2 inhibitors.

Cancer stem cells (CSCs) are an obstacle in cancer therapy and are a major cause of drug resistance, cancer recurrence, and metastasis. Available treatments, targeting proliferating cancer cells, are not effective in eliminating quiescent CSCs. Identification of CSC regulators will help design therapeutic strategies to sensitize drug-resistant CSCs for chemo-eradication. Here, we show that angiogenin and plexin-B2 regulate the stemness of prostate CSCs, and that inhibitors of angiogenin/plexin-B2 sensitize prostate CSCs to chemotherapy. Prostate CSCs capable of self-renewal, differentiation, and tumor initiation with a single cell inoculation were identified and shown to be regulated by angiogenin/plexin-B2 that promotes quiescence and self-renewal through 5S ribosomal RNA processing and generation of the bioactive 3'-end fragments of 5S ribosomal RNA, which suppress protein translation and restrict cell cycling. Monoclonal antibodies of angiogenin and plexin-B2 decrease the stemness of prostate CSCs and sensitize them to chemotherapeutic agents in vitro and in vivo.

Production and introduction of a novel immunotoxin based on engineered RNase A for inducing death to Her1-positive cell lines.

The present study was performed to design an immunotoxin consisting of engineered RNase A and scFv of Cetuximab. To accomplish this study goal, at first to evade RNase A from its inhibitors in the cytoplasm, six amino acids of RNase A were substituted, then the physicochemical features of engineered RNase A were assessed. To investigate the interaction between the engineered RNase A and the ribonuclease inhibitor, protein-protein docking was performed. After engineering the RNase A, it was theoretically conjugated with scFv of Cetuximab using a cleavable linker to produce scFv-engineered RNase A. Then, wild-RNase A (14 kD), engineered RNase A (14 kD) and scFv-engineered RNase A (42 kDa) were expressed in the BL21 (DE3) strain of Escherichia coli and purified by Ni-NTA columns. To confirm the expressed proteins, western blot analysis was performed. The functioning of wild-RNase A and engineered RNase A were investigated by RNA fragmentation assay. Finally, to evaluate the cytotoxicity of scFv-engineered RNase A, a dose-response cytotoxicity assay was performed on Her1-positive and Her1-negative cell lines. The results showed that engineered RNase A could maintain its structure and disulfide bonds and evade its inhibitor. Expression and purification were successfully conducted and both enzymes could degrade yeast RNA. The result of cytotoxicity showed that the engineered immunotoxin could induce cell death to Her1-positive cell lines with an IC50 of 50 nM. It appears that scFv-engineered RNase A can be a promising molecule for use.

Thioredoxin interacting protein (Txnip) forms redox sensitive high molecular weight nucleoprotein complexes.

Thioredoxin interacting protein (Txnip) is an α-arrestin protein that regulates pleiotropic biological responses. Txnip acts as a cancer suppressor and is a critical regulator of energy metabolism. To investigate molecular mechanisms involving Txnip, we searched for its protein binding partners using tandem affinity purification and proteomics analyses and identified several viable candidates, including HSP90, HSP70, and Prp31. We showed, by native PAGE, that Txnip is involved in the formation of high molecular weight complexes (1000-1300 kDa) in the nuclear fraction of cells treated with glucose and bortezomib. DTT treatment partly dissolved these high molecular weight complexes, suggesting that Txnip forms redox sensitive high-order nucleoprotein complexes. RNAse treatment slightly decreased the complex and RNA-seq showed differential expression of RNAs in the complex between Txnip protein overexpressing and control cells, indicating the involvement of RNAs in the complex. These results collectively provide a model whereby Txnip exerts its functions through multiple binding partners, forming transient higher-order complexes to regulate other signaling molecules.

Enzyme-Instructed Activation of Pro-protein Therapeutics In Vivo.

The selective and temporal control of protein activity in living cells provides a powerful tool to manipulate cellular function and to develop pro-protein therapeutics (PPT) for targeted therapy. In this work, we reported a facile but general chemical approach to design PPT by modulating protein activity in response to endogenous enzyme of disease cells, and its potential for targeted cancer therapy. We demonstrated that the chemical modification of a protein with quinone propionic acid (QPN), a ligand that could be reduced by tumor-cell-specific NAD(P)H dehydrogenase [quinone] 1 (NQO1), was reversible in the presence of NQO1. Importantly, the QPN-modified cytochrome c (Cyt c-QPN) and ribonuclease A (RNase A-QPN) showed NQO1-regulated protein activity in a highly selective manner. Furthermore, the intracellular delivery of RNase A-QPN using a novel type of lipid-based nanoparticles, and subsequent protein activation by cellular NQO1, selectively inhibit cancer cell growth in vitro and effectively suppress tumor growth in vivo. We believe that our approach increases the number of potentially useful chemical tools for reversibly controlling the structure and function of protein using a disease-cell-specific enzyme, opening opportunities in the study of dynamic biological processes and developing precise protein therapeutics.

Vascular regression precedes motor neuron loss in the FUS (1-359) ALS mouse model.

Amyotrophic lateral sclerosis (ALS) presents a poorly understood pathogenesis. Evidence from patients and mutant SOD1 mouse models suggests vascular damage may precede or aggravate motor dysfunction in ALS. We have previously shown angiogenin (ANG) treatment enhances motor neuron survival, delays motor dysfunction and prevents vascular regression in the SOD1G93A ALS model. However, the existence of vascular defects at different stages of disease progression remains to be established in other ALS models. Here, we assessed vascular integrity in vivo throughout different disease stages, and investigated whether ANG treatment reverses vascular regression and prolongs motor neuron survival in the FUS (1-359) mouse model of ALS. Lumbar spinal cord tissue was collected from FUS (1-359) and non-transgenic control mice at postnatal day (P)50, P90 and P120. We found a significant decrease in vascular network density in lumbar spinal cords from FUS (1-359) mice by day 90, at which point motor neuron numbers were unaffected. ANG treatment did not affect survival or counter vascular regression. Endogenous Ang1 and Vegf expression were unchanged at P50 and P90; however, we found a significant decrease in miRNA 126 at P50, indicating vascular integrity in FUS mice may be compromised via an alternative pathway. Our study demonstrates that vascular regression occurs before motor neuron degeneration in FUS (1-359) mice, and highlights that heterogeneity in responses to novel ALS therapeutics can already be detected in preclinical mouse models of ALS.This article has an associated First Person interview with the joint first authors of the paper.

Redox-Responsive Polymeric Nanocomplex for Delivery of Cytotoxic Protein and Chemotherapeutics.

Responsive delivery of anticancer proteins into cells is an emerging field in biological therapeutics. Currently, the delivery of proteins is highly compromised by multiple successive physiological barriers that reduce the therapeutic efficacy. Hence, there is a need to design a robust and sustainable nanocarrier to provide suitable protection of proteins and overcome the physiological barriers for better cellular accumulation. In this work, polyethylenimine (PEI) cross-linked by oxaliplatin(IV) prodrug (oxliPt(IV)) was used to fabricate a redox-responsive nanocomplex (PEI-oxliPt(IV)@RNBC/GOD) for the delivery of a reactive oxygen species-cleavable, reversibly caged RNase A protein (i.e., RNase A nitrophenylboronic conjugate, RNBC) and glucose oxidase (GOD) in order to realize efficient cancer treatment. The generation of hydrogen peroxide by GOD can uncage and restore the enzymatic activity of RNBC. On account of the responsiveness of the nanocomplex to highly reducing cellular environment, it would dissociate and release the protein and active oxaliplatin drug, causing cell death by both catalyzing RNA degradation and inhibiting DNA synthesis. As assessed by the RNA degradation assay, the activity of the encapsulated RNBC was recovered by the catalytic production of hydrogen peroxide from GOD and glucose substrate overexpressed in cancer cells. Monitoring of the changes in nanoparticle size confirmed that the nanocomplex could dissociate in the reducing environment, with the release of active oxaliplatin drug and protein. Confocal laser scanning microscopy (CLSM) and flow cytometry analysis revealed highly efficient accumulation of the nanocomplex as compared to free native proteins. In vitro cytotoxicity experiments using 4T1 cancer cells showed ∼80% cell killing efficacy, with highly efficient apoptosis induction. Assisted by the cationic polymeric carrier, it was evident from CLSM images that intracellular delivery of the therapeutic protein significantly depleted the RNA level. Thus, this work provides a promising platform for the delivery of therapeutic proteins and chemotherapeutic drugs for efficient cancer treatment.

Angiogenin activates the astrocytic Nrf2/antioxidant-response element pathway and thereby protects murine neurons from oxidative stress.

The angiogenin (ANG) gene is mutated frequently in individuals with amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. Delivering human ANG to mice that display ALS-like symptoms extends their lifespan and improves motor function. ANG is a secretory vertebrate RNase that enters neuronal cells and cleaves a subset of tRNAs, leading to the inhibition of translation initiation and the assembly of stress granules. Here, using murine neuronal and astrocytic cell lines, we find that ANG triggers the activation of the Nrf2 (nuclear factor erythroid 2-related factor 2) pathway, which provides a critical cellular defense against oxidative stress. This activation, which occurred in astrocytes but not in neurons, promoted the survival of proximal neurons that had oxidative injury. These findings extend the role of ANG as a neuroprotective agent and underscore its potential utility in ALS management.

Exploratory investigation of the anti-inflammatory effects of RNase A-treated Enterococcus faecalis strain EC-12.

We previously reported that the major component of Enterococcus faecalis strain EC-12 (EC-12) inducing production of Interleukin (IL)-12 in mouse/human immune cells was its own RNA. This study aimed to investigate if RNase A-treated EC-12 could also produce IL-10 and to evaluate the possible effects of IL-10 produced by RNase A-treated EC-12. Three experiments were conducted: (1) Assessment of the effect of RNase A-treated EC-12 on transcriptome profiles and biological pathways in human peripheral blood mononuclear cells; (2) Determination of cytokine concentration in its culture supernatants; and (3) Supplementation of RNase A-treated EC-12 (RN) to mice with dextran sodium sulfate-induced colitis. Treatment of EC-12 with RNase A inhibited inflammatory response including the potency to induce IL-12 production, while it did not affect IL-10 production (Experiment 1 and 2). Colitis symptoms were milder in RN than in PBS-supplemented controls (Experiment 3). RNase A-treated EC-12 likely became an anti-inflammatory agent primarily inducing IL-10 production.

Nano-Scaled Zeolitic Imidazole Framework-8 as an Efficient Carrier for the Intracellular Delivery of RNase A in Cancer Treatment.

BACKGROUND: Zeolitic imidazole framework-8 (ZIF-8) as an emerging platform has exhibited great potential in the protein delivery owing to its tunable chemical functionality. MATERIALS AND METHODS: ZIF-8 was employed as a carrier for the encapsulation and intracellular delivery of RNase A, aimed to achieve a rapid release of proteins in an acidic environment. The intracellular uptake of RNase A was studied by confocal laser scanning microscopy (CLSM), and the inhibition of cell proliferation after the delivery of RNase A was evaluated by MTT assay, Live/Dead staining, and TUNEL cell apoptosis analysis, using human lung adenocarcinoma cell line A549 as a model. The biocompatibility of RNase A@ZIF-8 nanoparticles was systematically detected through the hemolysis and cytotoxicity assay. RESULTS: The RNase A@ZIF-8 nanoparticles constructed by biomimetic mineralization could not only facilitate the encapsulation of protein molecules (protein loading: 13.4%) but also maintain the enzymatic activity and stability of RNase A. The CLSM images showed that RNase A@ZIF-8 nanoparticles could efficiently improve the intracellular uptake of RNase A. Moreover, RNase A@ZIF-8 nanoparticles could obviously inhibit the cell proliferation through the induction of cell apoptosis, with 31.3% of cell death at an RNase A concentration of 10 µg/mL. Finally, RNase A@ZIF-8 nanoparticles were elucidated to possess excellent biocompatibility, with hemolysis of <5% using the same concentration of RNase A@ZIF-8. CONCLUSION: ZIF-8 could be used as an effective carrier to deliver the therapeutic protein RNase A into the cytosol, which will be beneficial for improving the efficacy of cancer treatment.

Construction of Highly Stable Cytotoxic Nuclear-Directed Ribonucleases.

Ribonucleases are proteins whose use is promising in anticancer therapy. We have previously constructed different human pancreatic ribonuclease variants that are selectively cytotoxic for tumor cells by introducing a nuclear localization signal into their sequence. However, these modifications produced an important decrease in their stability compromising their behavior in vivo. Here, we show that we can significantly increase the thermal stability of these cytotoxic proteins by introducing additional disulfide bonds by site-directed mutagenesis. One of these variants increases its thermal stability by around 17 °C, without affecting its catalytic activity while maintaining the cytotoxic activity against tumor cells. We also show that the most stable variant is significantly more resistant to proteolysis when incubated with proteinase K or with human sera, suggesting that its half-live could be increased in vivo once administered.

Queuosine modification protects cognate tRNAs against ribonuclease cleavage.

Eukaryotic transfer RNAs (tRNA) contain on average 13 modifications that perform a wide range of roles in translation and in the generation of tRNA fragments that regulate gene expression. Queuosine (Q) modification occurs in the wobble anticodon position of tRNAs for amino acids His, Asn, Tyr, and Asp. In eukaryotes, Q modification is fully dependent on diet or on gut microbiome in multicellular organisms. Despite decades of study, cellular roles of Q modification remain to be fully elucidated. Here we show that in human cells, Q modification specifically protects its cognate tRNAHis and tRNAAsn against cleavage by ribonucleases. We generated cell lines that contain completely depleted or fully Q-modified tRNAs. Using these resources, we found that Q modification significantly reduces angiogenin cleavage of its cognate tRNAs in vitro. Q modification does not change the cellular abundance of the cognate full-length tRNAs, but alters the cellular content of their fragments in vivo in the absence and presence of stress. Our results provide a new biological aspect of Q modification and a mechanism of how Q modification alters small RNA pools in human cells.

Effects of VEGF-ANG-1-PLA nano-sustained release microspheres on proliferation and differentiation of ADSCs.

The improvement of fat graft viability might depend on the presence of multipotent resident adipose derived stem cells (ADSCs) which is the important component of stromal vascular fraction (SVF). Vascular endothelial growth factor (VEGF) and angiogenin-1 (Ang-1) are responsible for neovascularization. However, their half-life is too short to produce a biological effect. We thus investigated whether VEGF-ANG-1-polylactic acid (PLA) microspheres could enhance the angiogenic properties of ADSCs. PLA microspheres containing VEGF and ANG-1 were prepared by in vitro ultrasonic emulsification and characterized according to their encapsulation efficiency (EE), drug-loading rate (DL), particle size, and drug release. The systemic toxicity of empty loaded nanospheres (NPs) and the ability of these microspheres to promote the proliferation and differentiation of ADSCs were evaluated. The EE and DL were above 86% and 0.0288%, respectively [corrected].The drug release was completed after 20 days. Systemic toxicity was verified in ADSCs that received the unloaded NPs. It was observed that ADSCs treated with VEGF-ANG-1-PLA microspheres had an increase in the proliferation and the number of CD31 positive cells. ADSCs proliferation and differentiation toward endothelial cells (ECs) could be enhanced by the addition of VEGF-ANG-1-PLA nano-sustained release microspheres.