DUX4-induced HSATII transcription causes KDM2A/B-PRC1 nuclear foci and impairs DNA damage response.

Polycomb repressive complexes regulate developmental gene programs, promote DNA damage repair, and mediate pericentromeric satellite repeat repression. Expression of pericentromeric satellite repeats has been implicated in several cancers and diseases, including facioscapulohumeral dystrophy (FSHD). Here, we show that DUX4-mediated transcription of HSATII regions causes nuclear foci formation of KDM2A/B-PRC1 complexes, resulting in a global loss of PRC1-mediated monoubiquitination of histone H2A. Loss of PRC1-ubiquitin signaling severely impacts DNA damage response. Our data implicate DUX4-activation of HSATII and sequestration of KDM2A/B-PRC1 complexes as a mechanism of regulating epigenetic and DNA repair pathways.

Pharmacologic therapy for engraftment arrhythmia induced by transplantation of human cardiomyocytes.

Heart failure remains a significant cause of morbidity and mortality following myocardial infarction. Cardiac remuscularization with transplantation of human pluripotent stem cell-derived cardiomyocytes is a promising preclinical therapy to restore function. Recent large animal data, however, have revealed a significant risk of engraftment arrhythmia (EA). Although transient, the risk posed by EA presents a barrier to clinical translation. We hypothesized that clinically approved antiarrhythmic drugs can prevent EA-related mortality as well as suppress tachycardia and arrhythmia burden. This study uses a porcine model to provide proof-of-concept evidence that a combination of amiodarone and ivabradine can effectively suppress EA. None of the nine treated subjects experienced the primary endpoint of cardiac death, unstable EA, or heart failure compared with five out of eight (62.5%) in the control cohort (hazard ratio = 0.00; 95% confidence interval: 0-0.297; p = 0.002). Pharmacologic treatment of EA may be a viable strategy to improve safety and allow further clinical development of cardiac remuscularization therapy.

Design of 2'-O-methyl RNA and DNA double-stranded oligonucleotides: naturally-occurring nucleotide components with strong RNA interference gene expression inhibitory activity.

Double-stranded RNAs consisting of 21-nucleotide passenger and guide strands, known as small interfering RNAs (siRNAs), can be used for the identification of gene functions and the regulation of genes involved in disease for therapeutics. The difficulty with unmodified siRNAs lies in the chemical synthesis of RNA, its degradation by RNase, the immune response derived from natural RNA, and the off-target effects mediated by the passenger strand. In this study, asymmetrical 18 base-paired double-strand oligonucleotides comprised of alternately combined DNAs and 2'-O-methyl RNAs, denoted as MED-siRNA, were evaluated. These modified oligonucleotides showed high RNase resistance, a reduced immune response, a highly efficient cleavage of target mRNA with binding to Argonaute 2 (Ago2) via RNA interference, and the subsequent reduction of target protein expression. These findings suggest the possibility of alternatives to unmodified siRNAs with potential use in therapeutics.

Attenuation of pulmonary fibrosis in type I collagen-targeted reporter mice with ALK-5 inhibitors.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease, and consequently, effective antifibrotic drugs are strongly desired. Although we have previously reported a validated Col1a1-Luc Tg rat model for fibrosis, there are only a few mouse models that enable the evaluation of fibrosis in a short time period and with high sensitivity. Therefore, we generated a Col1a1-internal ribosome entry site (IRES)-Luc knock-in (KI) mouse in which the IRES-luciferase gene construct was inserted into the 3'-UTR of the type I collagen alpha 1 gene (Col1a1). There was a high correlation between luciferase activity and hydroxyproline content in the KI mice, which is similar to the result that we have previously reported for the Col1a1-Luc Tg rat model. In a bleomycin (BLM)-induced lung fibrosis model, luciferase activity in the lung showed a significant increase 3 days after BLM treatment, while only a slight increase was observed in the hydroxyproline content. An ALK-5 inhibitor-R-268712-was effective in inhibiting the luciferase activity in both the in vivo BLM-induced lung fibrosis model and in vitro primary mouse lung fibroblasts. This suggests that fibroblasts are the major collagen-producing cells in lung fibrosis. In human lung fibroblasts, TGF-ß stimulation induced α-smooth muscle actin as observed by immunostaining, suggesting that myofibroblast transdifferentiation (MTD) plays an important role in lung fibrosis. Together, these results indicated that ALK-5 inhibitors might affect lung fibrosis mainly via the inhibition of MTD. Thus, the Col1a1-IRES-Luc KI mouse might be useful for the evaluation of antifibrotic effects and their underlying mechanisms.

Human embryonic stem cell-derived cardiomyocytes restore function in infarcted hearts of non-human primates.

Pluripotent stem cell-derived cardiomyocyte grafts can remuscularize substantial amounts of infarcted myocardium and beat in synchrony with the heart, but in some settings cause ventricular arrhythmias. It is unknown whether human cardiomyocytes can restore cardiac function in a physiologically relevant large animal model. Here we show that transplantation of ∼750 million cryopreserved human embryonic stem cell-derived cardiomyocytes (hESC-CMs) enhances cardiac function in macaque monkeys with large myocardial infarctions. One month after hESC-CM transplantation, global left ventricular ejection fraction improved 10.6 ± 0.9% vs. 2.5 ± 0.8% in controls, and by 3 months there was an additional 12.4% improvement in treated vs. a 3.5% decline in controls. Grafts averaged 11.6% of infarct size, formed electromechanical junctions with the host heart, and by 3 months contained ∼99% ventricular myocytes. A subset of animals experienced graft-associated ventricular arrhythmias, shown by electrical mapping to originate from a point-source acting as an ectopic pacemaker. Our data demonstrate that remuscularization of the infarcted macaque heart with human myocardium provides durable improvement in left ventricular function.

Evaluation of the therapeutic index of a novel phosphodiesterase 4B-selective inhibitor over phosphodiesterase 4D in mice.

Phosphodiesterase 4 (PDE4) inhibitors have been developed for the treatment of pulmonary inflammatory diseases, but their clinical use was dose-limited by mainly gastric adverse effects. Recent studies suggested PDE4B-selective inhibitors over PDE4D are supposed to display a wider therapeutic index than subtype non-selective PDE4 inhibitors such as roflumilast. Compound A was identified as an orally active PDE4B-selective inhibitor over PDE4D both in humans (80-fold selective) and mice (29-fold selective). In this study, the therapeutic effects of compound A and roflumilast were evaluated on lipopolysaccaride (LPS) injection-induced plasma TNF-α elevation and on LPS inhalation-induced pulmonary neutrophilia in mice. The inhibitory effect on gastric emptying in mice was evaluated as a gastric adverse effect. The therapeutic index for TNF-α production (TI(TNF) = ID50 in gastric emptying / ID50 in LPS injection-induced plasma TNF-α elevation) of compound A was larger than roflumilast (9.0 and 0.2, respectively), whereas the therapeutic index for pulmonary neutrophilia (TI(Neu) = ID50 in gastric emptying / ID50 in LPS inhalation-induced pulmonary neutrophilia) of compound A was comparable to roflumilast (1.0 and 0.5, respectively). In conclusion, the TI(Neu) of compound A was not superior compared to that of roflumilast in spite of its high selectivity for PDE4B over PDE4D in mice.

Novel triple neurokinin receptor antagonist CS-003 inhibits respiratory disease models in guinea pigs.

Neurokinins are known to induce neurogenic inflammation related to respiratory diseases. The effects of CS-003 ([1-{2-[(2R)-(3,4-dichlorophenyl)-4-(3,4,5-trimethoxybenzoyl)morpholin-2-yl]ethyl}spiro[benzo[c]thiophene-1(3H),4'-piperidine]-(2S)-oxide hydrochloride]), a novel triple neurokinin receptor antagonist, on several respiratory disease models were evaluated in guinea pigs. As we have already shown that CS-003 is intravenously effective, we first determined if CS-003 was orally effective. CS-003 dose-dependently inhibited substance P-induced tracheal vascular hyperpermeability, neurokinin A- and neurokinin B-induced bronchoconstriction with ID(50) values of 3.6, 1.3 and 0.89 mg/kg (p.o.), respectively. CS-003 (10 mg/kg, p.o.) inhibited the number of coughs induced by capsaicin aerosol (P<0.01) and the antitussive effect was comparable to that of codeine. CS-003 (10 mg/kg, p.o.) also inhibited airway hyperresponsiveness to methacholine chloride in ovalbumin-induced asthma models (P<0.01), a milder one and a severer one. On the other hand, montelukast (10 mg/kg, p.o.), a leukotriene receptor antagonist, significantly inhibited the hyperresponsiveness only in the milder model (P<0.05). In an ovalbumin-induced rhinitis model, oral administration of CS-003 inhibited nasal blockade in a dose-dependent manner and the inhibitory effect was comparable to that of dexamethasone (10 mg/kg, p.o.). CS-003 (i.v.) also dose-dependently inhibited cigarette smoke-induced bronchoconstriction, tracheal vascular hyperpermeability and mucus secretion. These data show that CS-003, a potent orally active triple neurokinin receptor antagonist, may be useful for the treatment of respiratory diseases associated with neurokinins, such as allergic asthma, allergic rhinitis, chronic obstructive pulmonary disease and cough.