Non-coding RNA yREX3 from human extracellular vesicles exerts macrophage-mediated cardioprotection via a novel gene-methylating mechanism.

BACKGROUND AND AIMS: Extracellular vesicles (EVs) secreted by cardiosphere-derived cells exert immunomodulatory effects through the transmission of small non-coding RNAs. METHODS: The mechanism and role of yREX3, a small Y RNA abundant in EVs in myocardial injury, was investigated. RESULTS: yREX3 attenuates cardiac ischaemic injury by selective DNA methylation. Synthetic yREX3 encapsulated in lipid nanoparticles triggers broad transcriptomic changes in macrophages, localizes to the nucleus, and mediates epigenetic silencing of protein interacting with C kinase-1 (Pick1) through methylation of upstream CpG sites. Moreover, yREX3 interacts with polypyrimidine tract binding protein 3 (PTBP3) to methylate the Pick1 gene locus in a DNA methyltransferase-dependent manner. Suppression of Pick1 in macrophages potentiates Smad3 signalling and enhances efferocytosis, minimizing heart necrosis in rats with myocardial infarction. Adoptive transfer of Pick1-deficient macrophages recapitulates the cardioprotective effects of yREX3 in vivo. CONCLUSIONS: These findings highlight the role of a small Y RNA mined from EVs with a novel gene-methylating mechanism.

Fabrication and characterization of a new eco-friendly sulfonamide-chitosan derivative with enhanced antimicrobial and selective cytotoxicity properties.

Chitosan (CH) exhibits low antimicrobial activity. This study addresses this issue by modifying the chitosan with a sulfonamide derivative, 3-(4-(N,N-dimethylsulfonyl)phenyl)acrylic acid. The structure of the sulfonamide-chitosan derivative (DMS-CH) was confirmed using Fourier transform infrared spectroscopy and Nuclear magnetic resonance. The results of scanning electron microscopy, thermal gravimetric analysis, and X-ray diffraction indicated that the morphology changed to a porous nature, the thermal stability decreased, and the crystallinity increased in the DMS-CH derivative compared to chitosan, respectively. The degree of substitution was calculated from the elemental analysis data and was found to be moderate (42%). The modified chitosan exhibited enhanced antimicrobial properties at low concentrations, with a minimum inhibitory concentration (MIC) of 50 µg/mL observed for B. subtilis and P. aeruginosa, and a value of 25 µg/mL for S. aureus, E. coli, and C. albicans. In the case of native chitosan, the MIC values doubled or more, with 50 µg/mL recorded for E. coli and C. albicans and 100 µg/mL recorded for B. subtilis, S. aureus, and P. aeruginosa. Furthermore, toxicological examinations conducted on MCF-7 (breast adenocarcinoma) cell lines demonstrated that DMS-CH exhibited greater toxicity (IC50 = 225.47 µg/mL) than pure CH, while still maintaining significant safety limits against normal lung fibroblasts (WI-38). Collectively, these results suggest the potential use of the newly modified chitosan in biomedical applications.

Oral bioavailability of a noncoding RNA drug, TY1, that acts on macrophages.

All approved RNA therapeutics require parenteral delivery. Here we demonstrate an orally bioavailable formulation wherein synthetic noncoding (nc) RNA, packaged into lipid nanoparticles, is loaded into casein-chitosan (C2) micelles. We used the C2 formulation to deliver TY1, a 24-nucleotide synthetic ncRNA which targets the DNA damage response pathway in macrophages. C2-formulated TY1 (TY1C2) efficiently packages and protects TY1 against degradative enzymes. In healthy mice, oral TY1C2 was well-tolerated and nontoxic. Oral TY1C2 exhibited disease-modifying bioactivity in 2 models of tissue injury: 1) rat myocardial infarction, where a single oral dose of TY1C2 was cardioprotective, on par with intravenously-delivered TY1; and 2) mouse acute lung injury, where a single dose of TY1C2 attenuated pulmonary inflammation. Mechanistic dissection revealed that TY1C2 is not absorbed into the systemic circulation but is, instead, taken up by intestinal macrophages, namely those of the lamina propria and Peyer's patches. This route of absorption may rationalize why an antisense oligonucleotide against Factor VII, which acts on hepatocytes, is not effective when administered in the C2 formulation. Thus, some (but not all) ncRNA drugs are bioavailable when delivered by mouth. Oral RNA delivery and uptake, relying on uptake via the gastrointestinal immune system, has broad-ranging therapeutic implications.

A novel micropeptide, Slitharin, exerts cardioprotective effects in myocardial infarction.

PURPOSE: Micropeptides are an emerging class of proteins that play critical roles in cell signaling. Here, we describe the discovery of a novel micropeptide, dubbed slitharin (Slt), in conditioned media from Cardiosphere-derived cells (CDCs), a therapeutic cardiac stromal cell type. EXPERIMENTAL DESIGN: We performed mass spectrometry of peptide-enriched fractions from the conditioned media of CDCs and a therapeutically inert cell type (human dermal fibrobasts). We then evaluated the therapeutic capacity of the candidate peptide using an in vitro model of cardiomyocyte injury and a rat model of myocardial infarction. RESULTS: We identified a novel 24-amino acid micropeptide (dubbed Slitharin [Slt]) with a non-canonical leucine start codon, arising from long intergenic non-coding (LINC) RNA 2099. Neonatal rat ventricular myocytes (NRVMs) exposed to Slt were protected from hypoxic injury in vitro compared to a vehicle or scrambled control. Transcriptomic analysis of cardiomyocytes exposed to Slt reveals cytoprotective capacity, putatively through regulation of stress-induced MAPK-ERK. Slt also exerted cardioprotective effects in rats with myocardial infarction as shown by reduced infarct size 48 h post-injury. Conclusions and clinical relavance: Thus, Slt is a non-coding RNA-derived micropeptide, identified in the extracellular space, with a potential cardioprotective function.

Synthesis, characterization, and biological evaluation of new chitosan derivative bearing diphenyl pyrazole moiety.

This work reports the synthesis of a new pyrazole derivative by reacting 5-amino-1,3-diphenyl pyrazole with succinic anhydride and bearing the product chemically on the chitosan chains via amide linkage to achieve a new chitosan derivative (DPPS-CH). The prepared chitosan derivative was analyzed by IR, NMR, elemental analysis, XRD, TGA-DTG, and SEM. As compared with chitosan, DPPS-CH showed an amorphous and porous structure. Coats-Redfern results showed that the thermal activation energy for the first decomposition of DPPS-CH is 43.72 KJ mol-1 lower than that required for chitosan (88.32 KJ mol-1), indicating the accelerating effect of DPPS on the thermal decomposition of DPPS-CH. The DPPS-CH manifested a powerful wide spectrum antimicrobial potential against pathogenic gram-positive and gram-negative bacteria and Candida albicans at minute concentrations (MIC = 50 µg mL-1) compared to chitosan (MIC = 100 µg mL-1). The MTT assay proved the toxic properties of DPPS-CH against a cancer cell line (MCF-7) at a minute concentration (IC50 = 15.14 µg mL-1) while affecting normal cells (WI-38) at seven times this concentration (IC50 = 107.8 µg mL-1). According to the current findings, the chitosan derivative developed in this work appears to be a promising material for use in biological domains.

Systemic Delivery of Extracellular Vesicles Attenuates Atrial Fibrillation in Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Atrial fibrillation (AF) is a common comorbidity in heart failure with preserved ejection fraction (HFpEF) patients. To date, treatments for HFpEF-related AF have been limited to anti-arrhythmic drugs and ablation. Here we examined the effects of immortalized cardiosphere-derived extracellular vesicles (imCDCevs) in rats with HFpEF. OBJECTIVES: This study sought to investigate the mechanisms of AF in HFpEF and probe the potential therapeutic efficacy of imCDCevs in HFpEF-related AF. METHODS: Dahl salt-sensitive rats were fed a high-salt diet for 7 weeks to induce HFpEF and randomized to receive imCDCevs (n = 18) or vehicle intravenously (n = 14). Rats fed a normal-salt diet were used as control animals (n = 26). A comprehensive characterization of atrial remodeling was conducted using functional and molecular techniques. RESULTS: HFpEF-verified animals showed significantly higher AF inducibility (84%) compared with control animals (15%). These changes were associated with prolonged action potential duration, slowed conduction velocity (connexin 43 lateralization), and fibrotic remodeling in the left atrium of HFpEF compared with control animals. ImCDCevs reversed adverse electrical remodeling (restoration of action potential duration to control levels and reorganization of connexin 43) and reduced AF inducibility (33%). In addition, fibrosis, inflammation, and oxidative stress, which are major pathological AF drivers, were markedly attenuated in imCDCevs-treated animals. Importantly, these effects occurred without changes in blood pressure and diastolic function. CONCLUSIONS: Thus, imCDCevs attenuated adverse remodeling, and prevented AF in a rat model of HFpEF.

Biodistribution of unmodified cardiosphere-derived cell extracellular vesicles using single RNA tracing.

Extracellular vesicles (EVs) are potent signalling mediators. Although interest in EV translation is ever-increasing, development efforts are hampered by the inability to reliably assess the uptake of EVs and their RNA cargo. Here, we establish a novel qPCR-based method for the detection of unmodified EVS using an RNA Tracer (DUST). In this proof-of-concept study we use a human-specific Y RNA-derived small RNA (YsRNA) we dub "NT4" that is enriched in cardiosphere-derived cell small EVs (CDC-sEVs). The assay is robust, sensitive, and reproducible. Intravenously administered CDC-sEVs accumulated primarily in the heart on a per mg basis. Cardiac injury enhanced EV uptake in the heart, liver, and brain. Inhibition of EV docking by heparin suppressed uptake variably, while inhibition of endocytosis attenuated uptake in all organs. In vitro, EVs were uptaken more efficiently by macrophages, endothelial cells, and cardiac fibroblasts compared to cardiomyocytes. These findings demonstrate the utility of DUST to assess uptake of EVs in vivo and in vitro.

Synthesis and characterization of new functionalized chitosan and its antimicrobial and in-vitro release behavior from topical gel.

Recently, chitosan and its derivatives have been gaining more attention due to their high integration into various biomedical applications. Herein, a new chitosan derivative was prepared by linking the chitosan (Cs) with a novel heterocyclic compound, Benzoimidazolyl-thiadiazole (BzimTD) to form Cs-BzimTD. The synthesis of the new chitosan derivative was confirmed by Fourier-Transform Infrared (FT-IR) spectroscopy, proton nuclear magnetic resonance (1H NMR), thermogravimetric (TGA-DTG) analysis, elemental analysis, and UV-Visible spectrophotometer. Data showed the high efficacy of functionalized Cs-BzimTD to inhibit the growth of pathogenic microbes, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans, with inhibition zones of 15.3 ± 0.6 - 9.2 ± 0.3 mm. Also, Cs-BzimTD was applied in a topical gel formulation by using two different polymers, Carbopol 940 (CP) and Carboxymethyl Cellulose (CMC) to form three gel formulations: Cs-BzimTD-CP, Cs-BzimTD-CMC, and Cs-BzimTD-CP-CMC. The new gels were checked for physical appearance, viscosity, Cs-BzimTD release, pH, spread-ability, and drug content. The results showed that all formulations were clear, transparent, and homogeneous with non-irritant pH values for skin (6.4 - 6.8). The spread-ability was found in the range of 7.1 - 9.4 g.cm/s. The Cs-BzimTD-CP formula showed the maximal Cs-BzimTD content percentage (86.5%) and the Cs-BzimTD release varied from 89.9 to 81.6% after 8 h depending on the gel formulation, with a maximum release achieved for Cs-BzimTD-CMC.

New thiadiazole modified chitosan derivative to control the growth of human pathogenic microbes and cancer cell lines.

The emergence of multidrug-resistant microbes and the propagation of cancer cells are global health issues. The unique properties of chitosan and its derivatives make it an important candidate for therapeutic applications. Herein, a new thiadiazole derivative, 4-((5-(butylthio)-1,3,4-thiadiazol-2-yl) amino)-4-oxo butanoic acid (BuTD-COOH) was synthesized and used to modify the chitosan through amide linkages, forming a new thiadiazole chitosan derivative (BuTD-CH). The formation of thiadiazole and the chitosan derivative was confirmed by FT-IR, 1H/13C-NMR, GC-MS, TGA, Elemental analysis, and XPS. The BuTD-CH showed a high antimicrobial effect against human pathogens Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, and Candida albicans with low MIC values of 25-50 µg ml-1 compared to unmodified chitosan. The in-vitro cytotoxicity of BuTD-CH was evaluated against two cancer cell lines (MCF-7 and HepG2) and one normal cell (HFB4) using the MTT method. The newly synthesized derivatives showed high efficacy against cancerous cells and targeted them at low concentrations (IC50 was 178.9 ± 9.1 and 147.8 ± 10.5 µg ml-1 for MCF-7 and HepG2, respectively) compared with normal HFB4 cells (IC50 was 335.7 ± 11.4 µg ml-1). Thus, low concentrations of newly synthesized BuTD-CH could be safely used as an antimicrobial and pharmacological agent for inhibiting the growth of human pathogenic microbes and hepatocellular and adenocarcinoma therapy.

Restoration of hand function in a rat model of repair of brachial plexus injury.

The incurability of spinal cord injury and subcortical strokes is due to the inability of nerve fibres to regenerate. One of the clearest clinical situations where failure of regeneration leads to a permanent functional deficit is avulsion of the brachial plexus. In current practice, surgical re-implantation of avulsed spinal roots provides a degree of motor recovery, but the patients neither recover sensation nor the use of the hand. In the present rat study, we show that transplantation of cultured adult olfactory ensheathing cells restores the sensory input needed for a complex, goal-directed fore-paw function and re-establishes synaptic transmission to the spinal grey matter and cuneate nucleus by providing a bridge for regeneration of severed dorsal root fibres into the spinal cord. Success in a first application of human olfactory ensheathing cells in clinical brachial plexus injury would open the way to the wider field of brain and spinal cord injuries.

Extracellular Vesicles as Therapeutic Agents for Cardiac Fibrosis.

Heart disease remains an increasing major public health challenge in the United States and worldwide. A common end-organ feature in diseased hearts is myocardial fibrosis, which stiffens the heart and interferes with normal pump function, leading to pump failure. The development of cells for regenerative therapy has been met with many pitfalls on its path to clinical translation. Recognizing that regenerative cells secrete therapeutically bioactive vesicles has paved the way to circumvent many failures of cell therapy. In this review, we provide an overview of extracellular vesicles (EVs), with a focus on their utility as therapeutic agents for cardiac regeneration. We also highlight the engineering potential of EVs to enhance their therapeutic application.

Chitosan­g­maleic acid for effective removal of copper and nickel ions from their solutions.

Pollution of the environment associated with discharging the toxic heavy metals in water makes us focus the light to solve this problem. In continuation of our efforts, we aim in this work to utilize the graft copolymer (chitosan­g­maleic acid) to purify water from copper and nickel ions. The graft copolymer has been synthesized using gamma radiation and the grafting conditions have been optimized by studying the influence of acetic acid concentration (0.5-10% V/V), monomer concentration (5-17.5% w/v), chitosan concentration (0.25-2.5% w/v) and absorbed dose (0.5-5 kGy). FT-IR and TGA have been employed to characterize the graft copolymer. The metal ions uptake by the prepared graft copolymer was investigated and the influence of contact time, solution pH, polymer concentration, and metal ion concentration was studied. Adsorption kinetic models (pseudo-first-order, pseudo-second-order, and intra-particle diffusion equations) and adsorption isotherms (Langmuir, Freundlich, and Temkin equations) were also studied. It was found that the adsorption kinetics and isotherm agreed well with pseudo-second-order and Langmuir equations, respectively, indicating that the adsorption was chemisorption. The adsorption capacities of CTS­g­MA were 312.4 mg g-1 and 70.1 mg g-1 for Cu2+ and Ni2+, respectively. Effect of co-existence of other cationic ions on the adsorption capacity was also investigated.

Disease-modifying bioactivity of intravenous cardiosphere-derived cells and exosomes in mdx mice.

Dystrophin deficiency leads to progressive muscle degeneration in Duchenne muscular dystrophy (DMD) patients. No known cure exists, and standard care relies on the use of antiinflammatory steroids, which are associated with side effects that complicate long-term use. Here, we report that a single intravenous dose of clinical-stage cardiac stromal cells, called cardiosphere-derived cells (CDCs), improves the dystrophic phenotype in mdx mice. CDCs augment cardiac and skeletal muscle function, partially reverse established heart damage, and boost the regenerative capacity of skeletal muscle. We further demonstrate that CDCs work by secreting exosomes, which normalize gene expression at the transcriptome level, and alter cell signaling and biological processes in mdx hearts and skeletal muscle. The work reported here motivated the ongoing HOPE-2 clinical trial of systemic CDC delivery to DMD patients, and identifies exosomes as next-generation cell-free therapeutic candidates for DMD.

Augmenting canonical Wnt signalling in therapeutically inert cells converts them into therapeutically potent exosome factories.

Cardiosphere-derived cells are therapeutic candidates with disease-modifying bioactivity, but their variable potency has complicated their clinical translation. Transcriptomic analyses of cardiosphere-derived cells from human donors have revealed that their therapeutic potency correlates with Wnt/ß-catenin signalling and with ß-catenin protein levels. Here, we show that skin fibroblasts engineered to overexpress ß-catenin and the transcription factor Gata4 become immortal and therapeutically potent. Transplantation of the engineered fibroblasts into a mouse model of acute myocardial infarction led to improved cardiac function and mouse survival, and in the mdx mouse model of Duchenne muscular dystrophy, exosomes secreted by the engineered fibroblasts improved exercise capacity and reduced skeletal-muscle fibrosis. We also demonstrate that exosomes from high-potency cardiosphere-derived cells exhibit enhanced levels of miR-92a (a known potentiator of the Wnt/ß-catenin pathway), and that they activate cardioprotective bone-morphogenetic-protein signalling in cardiomyocytes. Our findings show that the modulation of canonical Wnt signalling can turn therapeutically inert mammalian cells into immortal exosome factories for cell-free therapies.

Basilar impression and osteogenesis imperfecta: a 21-year retrospective review of outcomes in 20 patients.

OBJECT: Basilar impression (BI) secondary to osteogenesis imperfecta (OI) is a rare but debilitating condition that is often progressive unless it is halted. More recently, ventral decompression surgery has been advocated for this condition. This study is a retrospective review of the 21-year experience of ventral decompression surgery and dorsal occipitocervical fixation in patients with BI secondary to OI and is the largest patient series reported to date. METHODS: Twenty patients treated between 1982 and 2003 by the senior author at the authors' institution were included in this study. All patients underwent ventral decompression surgery followed by dorsal craniocervical stabilization. Patients were followed up for a median of 10 years. RESULTS: There were no intraoperative or perioperative deaths. Postoperatively, 16 of 20 (80%) patients showed objective improvement or maintained their good preoperative level of function. After surgery, of the 15 patients admitted with Karnofsky Performance Scale (KPS) scores of 70% or less, 11 improved, two remained unchanged, one patient's condition deteriorated, and one patient died of an unrelated cause. Of five patients admitted with a KPS score of 80% or greater, no patient's condition deteriorated in the short- and midterm period, but one patient had recurrence 15 years after surgery. At the end of follow-up, 25% of the patients had recurrence of brainstem compression symptoms or had died, and 15% showed no improvement after surgery. All of the remaining patients (60%) had sustained a long-term benefit from surgery. CONCLUSIONS: Aggressive ventral decompression surgery and dorsal stabilization for patients with BI secondary to OI can not only halt disease progression but can also produce a good and sustainable long-term functional outcome, even in those patients who present as severely symptomatic. Patients who presented early with minor symptoms had good long-term outcomes.

Concise Review: Developing Best-Practice Models for the Therapeutic Use of Extracellular Vesicles.

Growing interest in extracellular vesicles (EVs, including exosomes and microvesicles) as therapeutic entities, particularly in stem cell-related approaches, has underlined the need for standardization and coordination of development efforts. Members of the International Society for Extracellular Vesicles and the Society for Clinical Research and Translation of Extracellular Vesicles Singapore convened a Workshop on this topic to discuss the opportunities and challenges associated with development of EV-based therapeutics at the preclinical and clinical levels. This review outlines topic-specific action items that, if addressed, will enhance the development of best-practice models for EV therapies. Stem Cells Translational Medicine 2017;6:1730-1739.

Permanent loss of fore-paw grasping requires complete deprivation of afferent input from a minimum of four dorsal roots of the rat brachial plexus.

Dorsal roots from the 6th cervical to the 1st thoracic segment were sectioned flush with the surface of the spinal cord on one side. For 3 weeks before and 8 weeks after surgery the rats were filmed once a week during two successive climbs up a 1 m grid. Before surgery the fore-paws of normal rats grasped the grid bar for a mean of 7.0+/-0.1 times per climb. After complete section of C6 to T1 dorsal roots on one side there was a major deficit in the ipsilateral fore-paw in locating the grid bars, and grasping was almost totally abolished (mean of 0.1+/-0.06 grasps per climb). The failure of the rats to locate or to grasp the bars persisted unchanged for the entire test period. Rats with section of C6 to C8, but sparing T1, showed a similar but milder pattern of deficit. Section of any two adjacent cervical roots caused only minor deficits. Section of any single root alone caused no detectable deficit in climbing. The consistent loss of grasping after section of the 4 dorsal roots from C6 to T1 provides a promising model for assessing putative regenerative therapies.