Autophagic degradation of HAS2 in endothelial cells: A novel mechanism to regulate angiogenesis

Hyaluronan plays a key role in regulating inflammation and tumor angiogenesis. Of the three transmembrane hyaluronan synthases, HAS2 is the main pro-angiogenic enzyme responsible for excessive hyaluronan production. We discovered that HAS2 was degraded in vascular endothelial cells via autophagy evoked by nutrient deprivation, mTOR inhibition, or pro-autophagic proteoglycan fragments endorepellin and endostatin. Using live-cell and super-resolution confocal microscopy, we found that protracted autophagy evoked a dynamic interaction between HAS2 and ATG9A, a key transmembrane autophagic protein. This regulatory axis of HAS2 degradation occurred in various cell types and species and in vivo upon nutrient deprivation. Inhibiting in vivo autophagic flux via chloroquine showed increased levels of HAS2 in the heart and aorta. Functionally, autophagic induction via endorepellin or mTOR inhibition markedly suppressed extracellular hyaluronan production in vascular endothelial cells and inhibited ex vivo angiogenic sprouting. Thus, we propose autophagy as a novel catabolic mechanism regulating hyaluronan production in endothelial cells and demonstrate a new link between autophagy and angiogenesis that could lead to potential therapeutic modalities for angiogenesis.     

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.     

3D iPSC modeling of the retinal pigment epithelium-choriocapillaris complex identifies factors involved in the pathology of macular degeneration

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Simple production of resilin-like protein hydrogels using the Brevibacillus secretory expression system and column-free purification

Resilin, an insect structural protein, has excellent flexibility, photocrosslinking properties, and temperature responsiveness. Recombinant resilin-like proteins (RLPs) can be fabricated into three-dimensional (3D) structures for use as cell culture substrates and highly elastic materials. A simplified, high-yielding production process for RLPs is required for their widespread application. This study proposes a simple production process combining extracellular expression using Brevibacillus choshinensis (B. choshinensis) and rapid column-free purification. Extracellular production was tested using four representative signal peptides; B. choshinensis was found to efficiently secrete Rec1, an RLP derived from Drosophila melanogaster, regardless of the type of signal peptide. However, it was suggested that Rec1 is altered by an increase in the pH of the culture medium associated with prolonged incubation. Production in a jar fermentor with controllable pH yielded 530 mg Rec1 per liter of culture medium, which is superior to productivity using other hosts. The secreted Rec1 was purified from the culture supernatant via (NH₄)₂SO₄ and ethanol precipitations, and the purified Rec1 was applied to ring-shaped 3D hydrogels. These results indicate that the combination of secretory production using B. choshinensis and column-free purification can accelerate the further application of RLPs.     

Carboxymethyl cellulose–alginate interpenetrating hydroxy ethyl methacrylate crosslinked polyvinyl alcohol reinforced hybrid hydrogel templates with improved biological performance for cardiac tissue engineering

Cardiac tissue engineering is an emerging approach for cardiac regeneration utilizing the inherent healing responses elicited by the surviving heart using biomaterial templates. In this study, we aimed to develop hydrogel scaffolds for cardiac tissue regeneration following myocardial infarction (MI). Two superabsorbent hydrogels, CAHA2A and CAHA2AP, were developed employing interpenetration chemistry. CAHA2A was constituted with alginate, carboxymethyl cellulose, (hydroxyethyl) methacrylate, and acrylic acid, where CAHA2AP was prepared by interpenetrated CAHA2A with polyvinyl alcohol. Both hydrogels displayed superior physiochemical characteristics, as determined by attenuated total reflection infrared spectroscopy spectral analysis, differential scanning calorimetry measurements, tensile testing, contact angle, water profiling, dye release, and conductivity. In vitro degradation of the hydrogels displayed acceptable weight composure and pH changes. Both hydrogels were hemocompatible, and biocompatible as evidenced by direct contact and MTT assays. The hydrogels promoted anterograde and retrograde migration as determined by the z-stack analysis using H9c2 cells grown with both gels. Additionally, the coculture of the hydrogels with swine epicardial adipose tissue cells and cardiac fibroblasts resulted in synchronous growth without any toxicity. Also, both hydrogels facilitated the production of extracellular matrix by the H9c2 cells. Overall, the findings support an appreciable in vitro performance of both hydrogels for cardiac tissue engineering applications.     

Synthesis of Poly(N-isopropylacrylamide) Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability

Janus microparticles are compartmentalized particles with differing molecular structures and/or functionality on each of their two sides. Because of this unique property, Janus microparticles have been recognized as a new class of materials, thereby attracting a great deal of attention from various research fields. The versatility of these microparticles has been exemplified through their uses as building blocks for self-assembly, electrically responsive actuators, emulsifiers for painting and cosmetics, and carriers for drug delivery. This study introduces a detailed protocol that explicitly describes a synthetic method for designing novel Janus microhydrogels composed of a single base material, poly(N-isopropylacrylamide) (PNIPAAm). Janus microdroplets are firstly generated via a hydrodynamic focusing microfluidic device (HFMD) based on the separation of a supersaturated aqueous NIPAAm monomer solution and subsequently polymerized through exposure to UV irradiation. The resulting Janus microhydrogels were found to be entirely composed of the same base material, featured an easily identifiable compartmentalized morphology, and exhibited anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability. We believe that the proposed method introduces a novel hydrogel platform with the potential for advanced synthesis of multi-functional Janus microhydrogels.     

Chitosan hydrogels containing liposomes and cubosomes as particulate sustained release vaccine delivery systems

Sustained release depot systems have been widely investigated for their potential to improve the efficacy of subunit vaccines and reduce the requirement for boosting. The present study aimed to further enhance the immunogenicity of a sustained release vaccine by combining a depot formulation with a particulate antigen delivery system. Sustained release of the model subunit antigen, ovalbumin (OVA), was observed in vivo from chitosan thermogel-based formulations containing cationic, nanosized liposomes loaded with OVA and the immunopotentiator, Quil A (QA). Such formulations demonstrated the ability to induce cluster of differentiation (CD)8⁺ and CD4⁺ T-cell proliferation and interferon (IFN)-γ production, as well as the production of OVA-specific antibody. However, gel-incorporated liposomes showed evidence of instability and similar in vivo immune responses to liposomes in gel formulations were induced by gel-based systems loaded with soluble OVA and QA. The immunogenicity of chitosan thermogels containing cubosomes, a more stable lipidic particulate system, was therefore examined. Similarly, all gel-based formulations produced comparable effector immune responses in experimental mice, irrespective of whether the antigen and immunopotentiator were present in gels within cubosomes or in a soluble form. This work demonstrates the potential for sustained release thermogelling systems and highlights the importance of matching the physicochemical and immunological properties of the particulate system to that of the depot.     

Photodegradation as a mechanism for controlled drug delivery

A drug‐releasing model compound based on photosensitive acrylated ortho‐nitrobenzylether (o‐NBE) moiety and fluorescein was synthesized to demonstrate photolysis as a mechanism for drug release. Release of this model compound from a hydrogel network can be controlled with light intensity (5–20 mW/cm²), exposure duration (0–20 min) and wavelength (365, 405, 436 nm). Due to the high molar absorptivity of the compound (5,984 M^?1 cm^?1), light attenuation is significant in this system. Light attenuation can be used to self‐limit the dosing from a hydrogel, and allow subsequent release from the drug reservoir after equilibration, or attenuation can be utilized to create a chemical gradient within the hydrogel. A model of photodegradation that uses an integrated form of Beer–Lambert's law quantitatively predicts release from hydrophilic hydrogels with low crosslink density, but fails to quantitatively predict release from more hydrophobic systems, presumably due to partitioning of the hydrophobic model compound in the hydrogel. In contrast to other mechanisms of release (enzymolysis, hydrolysis), photolysis provides real‐time on demand control over drug release along with the unique ability to create chemical gradients within the hydrogel. Biotechnol. Bioeng. 2010;107: 1012–1019. © 2010 Wiley Periodicals, Inc.