Histone demethylase KDM5 regulates cardiomyocyte maturation by promoting fatty acid oxidation, oxidative phosphorylation, and myofibrillar organization.

AIMS: Human pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a platform to identify and characterize factors that regulate the maturation of CMs. The transition from an immature foetal to an adult CM state entails coordinated regulation of the expression of genes involved in myofibril formation and oxidative phosphorylation (OXPHOS) among others. Lysine demethylase 5 (KDM5) specifically demethylates H3K4me1/2/3 and has emerged as potential regulators of expression of genes involved in cardiac development and mitochondrial function. The purpose of this study is to determine the role of KDM5 in iPSC-CM maturation. METHODS AND RESULTS: KDM5A, B, and C proteins were mainly expressed in the early post-natal stages, and their expressions were progressively downregulated in the post-natal CMs and were absent in adult hearts and CMs. In contrast, KDM5 proteins were persistently expressed in the iPSC-CMs up to 60 days after the induction of myogenic differentiation, consistent with the immaturity of these cells. Inhibition of KDM5 by KDM5-C70 -a pan-KDM5 inhibitor, induced differential expression of 2372 genes, including upregulation of genes involved in fatty acid oxidation (FAO), OXPHOS, and myogenesis in the iPSC-CMs. Likewise, genome-wide profiling of H3K4me3 binding sites by the cleavage under targets and release using nuclease assay showed enriched of the H3K4me3 peaks at the promoter regions of genes encoding FAO, OXPHOS, and sarcomere proteins. Consistent with the chromatin and gene expression data, KDM5 inhibition increased the expression of multiple sarcomere proteins and enhanced myofibrillar organization. Furthermore, inhibition of KDM5 increased H3K4me3 deposits at the promoter region of the ESRRA gene and increased its RNA and protein levels. Knockdown of ESRRA in KDM5-C70-treated iPSC-CM suppressed expression of a subset of the KDM5 targets. In conjunction with changes in gene expression, KDM5 inhibition increased oxygen consumption rate and contractility in iPSC-CMs. CONCLUSION: KDM5 inhibition enhances maturation of iPSC-CMs by epigenetically upregulating the expressions of OXPHOS, FAO, and sarcomere genes and enhancing myofibril organization and mitochondrial function.

Strategies for overcoming protein and peptide instability in biodegradable drug delivery systems.

The global pharmaceutical market has recently shifted its focus from small molecule drugs to peptide, protein, and nucleic acid drugs, which now comprise a majority of the top-selling pharmaceutical products on the market. Although these biologics often offer improved drug specificity, new mechanisms of action, and/or enhanced efficacy, they also present new challenges, including an increased potential for degradation and a need for frequent administration via more invasive administration routes, which can limit patient access, patient adherence, and ultimately the clinical impact of these drugs. Controlled-release systems have the potential to mitigate these challenges by offering superior control over in vivo drug levels, localizing these drugs to tissues of interest (e.g., tumors), and reducing administration frequency. Unfortunately, adapting controlled-release devices to release biologics has proven difficult due to the poor stability of biologics. In this review, we summarize the current state of controlled-release peptides and proteins, discuss existing techniques used to stabilize these drugs through encapsulation, storage, and in vivo release, and provide perspective on the most promising opportunities for the clinical translation of controlled-release peptides and proteins.

Oxygen Ion Implantation Improving Cell Adhesion on Titanium Surfaces through Increased Attraction of Fibronectin PHSRN Domain.

Mechanistic understanding of fibronectin (FN) adsorption which determines cell adhesion on cell-implant interfaces is significant for improving the osteoconduction and soft-tissue healing of implants. Here, it is shown that the adsorption behavior of FN on the titanium oxide surface (TiO2 ) is highly relative to its Pro-His-Ser-Arg-Asn (PHSRN) peptide. FN lacking PHSRN fails to bind to surfaces, resulting in inhibited cell adhesion and spreading. Molecular dynamics simulation shows higher affinity and greater adsorption energy of PHSRN peptide with TiO2 surface due to the stronger hydrogen bonds formed by the serine and arginine residues with O ion of the substrate. Finally, by increasing O content in TiO2 surfaces through O ion-beam implantation, improving the cell adhesion, cell differentiation, and the subsequent biomineralization on titanium implant is realized. This study reveals the vital role of PHSRN in FN-mediated cell adhesion on implant surfaces, providing a promising new target for further tissue integration and implant success.

Size-Confined Effects of Nanostructures on Fibronectin-Induced Macrophage Inflammation on Titanium Implants.

Macrophage activation determines the fate of biomaterials implantation. Though researches have shown that fibronectin (FN) is highly involved in integrin-induced macrophage activation on biomaterials, the mechanism of how nanosized structure affects macrophage behavior is still unknown. Here, titanium dioxide nanotube structures with different sizes are fabricated to investigate the effects of nanostructure on macrophage activation. Compared with larger sized nanotubes and smooth surface, 30 nm nanotubes exhibit considerable lesser pro-inflammatory properties on macrophage differentiation. Confocal protein observation and molecular dynamics simulation show that FN displays conformation changes on different nanotubes in a feature of "size-confined," which causes the hiding of Arg-Gly-Asp (RGD) domain on other surfaces. The matching size of nanotube with FN allows the maximum exposure of RGD on 30 nm nanotubes, activating integrin-mediated focal adhesion kinase (FAK)-phosphatidylinositol-3 kinase γ (PI3Kγ) pathway to inhibit nuclear factor kappa B (NF-κB) signaling. In conclusion, this study explains the mechanism of nanostructural-biological signaling transduction in protein and molecular levels, as well as proposes a promising strategy for surface modification to regulate immune responses on bioimplants.

Photoactivated Trifunctional Platinum Nanobiotics for Precise Synergism of Multiple Antibacterial Modes.

Integrating multiple strategies of antibacterial mechanisms into one has been proven to have tremendous promise for improving antimicrobial efficiency. Hence, dual-valent platinum nanoparticles (dvPtNPs) with a zero-valent platinum core (Pt0 ) and bi-valent platinum shell (Pt2+ ions), combining photothermal and photodynamic therapy, together with "chemotherapy," emerge as spatiotemporally light-activatable platinum nano-antibiotics. Under near-infrared (NIR) exposure, the multiple antibacterial modes of dvPtNPs are triggered. The Pt0 core reveals significant hyperthermia via effective photothermal conversion while an immediate release of chemotherapeutic Pt2+ ions occurs through hyperthermia-initiated destabilization of metallic interactions, together with reactive oxygen species (ROS) level increase, thereby resulting in synergistic antibacterial effects. The precise cooperative effects between photothermal, photodynamic, and Pt2+ antibacterial effects are achieved on both Gram-negative Escherichia coli and Gram-positive methicillin-resistant Staphylococcus aureus, where bacterial viability and colony-forming units are significantly reduced. Moreover, similar results are observed in mice subcutaneous abscess models. Significantly, after NIR treatment, dvPtNP exhibits a more robust bacteria-killing efficiency than other PtNP groups, owing to its integration of dramatic damage to the bacterial membrane and DNA, and alteration to ATP and ROS metabolism. This study broadens the avenues for designing and synthesizing antibacterial materials with higher efficiency.

Near-Infrared Light-Sensitive Nano Neuro-Immune Blocker Capsule Relieves Pain and Enhances the Innate Immune Response for Necrotizing Infection.

Sensory neurons promote profound suppressive effects on neutrophils during Streptococcus pyogenes infection and contribute to the pathogenesis of necrotizing infection ("flesh-eating disease"). Thus, the development of new antibacterial agents for necrotizing infection is promising because of the clear streptococcal neuro-immune communication. Herein, based on the immune escape membrane exterior and competitive membrane functions of the glioma cell membrane, a novel nano neuro-immune blocker capsule was designed to prevent neuronal activation and improve neutrophil immune responses for necrotizing infection. These nano neuro-immune blockers could neutralize streptolysin S, suppress neuron pain conduction and calcitonin gene-related peptide release, and recruit neutrophils to the infection site, providing a strong therapeutic effect against necrotizing infection. Furthermore, nano neuro-immune blockers could serve as an effective inflammatory regulator and antibacterial agent via photothermal effects under near-infrared irradiation. In the Streptococcus pyogenes-induced necrotizing fasciitis mouse model, nano neuro-immune blockers showed significant therapeutic efficacy by ameliorating sensitivity to pain and promoting the antibacterial effect of neutrophils.

Pretreated Macrophage-Membrane-Coated Gold Nanocages for Precise Drug Delivery for Treatment of Bacterial Infections.

Pathogenic bacterial infections and drug resistance make it urgent to develop new antibacterial agents with targeted delivery. Here, a new targeting delivery nanosystem is designed based on the potential interaction between bacterial recognizing receptors on macrophage membranes and distinct pathogen-associated molecular patterns in bacteria. Interestingly, the expression of recognizing receptors on macrophage membranes increases significantly when cultured with specific bacteria. Therefore, by coating pretreated macrophage membrane onto the surface of a gold-silver nanocage (GSNC), the nanosystem targets bacteria more efficiently. Previously, it has been shown that GSNC alone can serve as an effective antibacterial agent owing to its photothermal effect under near-infrared (NIR) laser irradiation. Furthermore, the nanocage can be utilized as a delivery vehicle for antibacterial drugs since the gold-silver nanocage presents a hollow interior and porous wall structure. With significantly improved bacterial adherence, the Sa-M-GSNC nanosystem, developed within this study, is effectively delivered and retained at the infection site both via local or systemic injections; the system also shows greatly prolonged blood circulation time and excellent biocompatibility. The present work described here is the first to utilize bacterial pretreated macrophage membrane receptors in a nanosystem to achieve specific bacterial-targeted delivery, and provides inspiration for future therapy based on this concept.

Deproteinized bovine bone matrix induces osteoblast differentiation via macrophage polarization.

Bone grafts are widely used in bone regeneration to increase the speed and quality of new bone formation. While they are routinely characterized based on their biocompatible and bioactive properties, they also exert a profound impact on host immune responses, which in turn can display a significant effect on the healing and repair process. In this study, we investigated the role of macrophage behavior on deproteinized bovine bone matrix (DBBM, BioOss) to investigate their impact on creating either a pro- or anti-inflammatory microenvironment for tissue integration. RT-PCR and immunofluorescence staining results demonstrated the ability for RAW 264.7 cells to polarize toward M2 wound-healing macrophages in response to DBBM and positive control (IL-4). Interestingly, significantly higher expression of interleukin-10 and higher number of multinucleated giant cells (MNGCs) was observed in the DBBM group. Thereafter, conditioned media (CM) from macrophages cultured with DBBM seeded with MC3T3-E1 cells demonstrated a marked increase in osteoblast differentiation. Noteworthy, this effect was reversed by blocking IL10 with addition of IL10 antibody to CM from the DBBM macrophages. Furthermore, the use of dendritic cell specific transmembrane protein (DC-STAMP)-knockout to inhibit MNGC formation in the DBBM group resulted in a significant reduction in osteoblast differentiation, indication a pivotal role for MNGCs in biomaterials-induced osteogenesis. The results from this study indicate convincingly that the immune response of macrophages towards DBBM has a potent effect on osteoblast differentiation. Furthermore, DBBM promoted macrophage fusion and polarization towards an M2 wound-healing phenotype, further created a microenvironment favoring biomaterial-induced osteogenesis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1236-1246, 2018.

A Difunctional Regeneration Scaffold for Knee Repair based on Aptamer-Directed Cell Recruitment.

To solve the challenge of poor knee repair, an aptamer-bilayer scaffold is designed for autologous mesenchymal stem cell (MSC) recruitment and osteochondral regeneration. The scaffold can efficiently recruit MSCs to the defect and induce the directional differentiation of MSCs, thus successfully achieving simultaneous regeneration of cartilage and bone in the knee joint.