Hydrogel dressings with intrinsic antibiofilm and antioxidative dual functionalities accelerate infected diabetic wound healing.

Chronic wounds are often infected with biofilm bacteria and characterized by high oxidative stress. Current dressings that promote chronic wound healing either require additional processes such as photothermal irradiation or leave behind gross amounts of undesirable residues. We report a dual-functionality hydrogel dressing with intrinsic antibiofilm and antioxidative properties that are synergistic and low-leaching. The hydrogel is a crosslinked network with tethered antibacterial cationic polyimidazolium and antioxidative N-acetylcysteine. In a murine diabetic wound model, the hydrogel accelerates the closure of wounds infected with methicillin-resistant Staphylococcus aureus or carbapenem-resistant Pseudomonas aeruginosa biofilm. Furthermore, a three-dimensional ex vivo human skin equivalent model shows that N-acetylcysteine promotes the keratinocyte differentiation and accelerates the re-epithelialization process. Our hydrogel dressing can be made into different formats for the healing of both flat and deep infected chronic wounds without contamination of the wound or needing other modalities such as photothermal irradiation.

Attenuating Epithelial-to-Mesenchymal Transition in Cancer through Angiopoietin-Like 4 Inhibition in a 3D Tumor Microenvironment Model.

Epithelial-to-mesenchymal transition (EMT) plays a crucial role in metastatic cancer progression, and current research, which relies heavily on 2D monolayer cultures, falls short in recapitulating the complexity of a 3D tumor microenvironment. To address this limitation, a transcriptomic meta-analysis is conducted on diverse cancer types undergoing EMT in 2D and 3D cultures. It is found that mechanotransduction is elevated in 3D cultures and is further intensified during EMT, but not during 2D EMT. This analysis reveals a distinct 3D EMT gene signature, characterized by extracellular matrix remodeling coordinated by angiopoietin-like 4 (Angptl4) along with other canonical EMT regulators. Utilizing hydrogel-based 3D matrices with adjustable mechanical forces, 3D cancer cultures are established at varying physiological stiffness levels. A YAP:EGR-1 mediated up-regulation of Angptl4 expression is observed, accompanied by an upregulation of mesenchymal markers, at higher stiffness during cancer EMT. Suppression of Angptl4 using antisense oligonucleotides or anti-cAngptl4 antibodies leads to a dose-dependent abolishment of EMT-mediated chemoresistance and tumor self-organization in 3D, ultimately resulting in diminished metastatic potential and stunted growth of tumor xenografts. This unique programmable 3D cancer cultures simulate stiffness levels in the tumor microenvironment and unveil Angptl4 as a promising therapeutic target to inhibit EMT and impede cancer progression.

YWHAG Deficiency Disrupts the EMT-Associated Network to Induce Oxidative Cell Death and Prevent Metastasis.

Metastasis involves epithelial-to-mesenchymal transition (EMT), a process that is regulated by complex gene networks, where their deliberate disruption may yield a promising outcome. However, little is known about mechanisms that coordinate these metastasis-associated networks. To address this gap, hub genes with broad engagement across various human cancers by analyzing the transcriptomes of different cancer cell types undergoing EMT are identified. The oncogenic signaling adaptor protein tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (YWHAG) is ranked top for its clinical relevance and impact. The cellular kinome and transcriptome data are surveyed to construct the regulome of YWHAG, revealing stress responses and metabolic processes during cancer EMT. It is demonstrated that a YWHAG-dependent cytoprotective mechanism in the regulome is embedded in EMT-associated networks to protect cancer cells from oxidative catastrophe through enhanced autophagy during EMT. YWHAG deficiency results in a rapid accumulation of reactive oxygen species (ROS), delayed EMT, and cell death. Tumor allografts show that metastasis potential and overall survival time are correlated with the YWHAG expression level of cancer cell lines. Metastasized tumors have higher expression of YWHAG and autophagy-related genes than primary tumors. Silencing YWHAG diminishes primary tumor volumes, prevents metastasis, and prolongs the median survival period of the mice.

Microbial Dysregulation of the Gut-Lung Axis in Bronchiectasis.

Rationale: Emerging data support the existence of a microbial "gut-lung" axis that remains unexplored in bronchiectasis. Methods: Prospective and concurrent sampling of gut (stool) and lung (sputum) was performed in a cohort of n = 57 individuals with bronchiectasis and subjected to bacteriome (16S rRNA) and mycobiome (18S Internal Transcribed Spacer) sequencing (total, 228 microbiomes). Shotgun metagenomics was performed in a subset (n = 15; 30 microbiomes). Data from gut and lung compartments were integrated by weighted similarity network fusion, clustered, and subjected to co-occurrence analysis to evaluate gut-lung networks. Murine experiments were undertaken to validate specific Pseudomonas-driven gut-lung interactions. Results: Microbial communities in stable bronchiectasis demonstrate a significant gut-lung interaction. Multibiome integration followed by unsupervised clustering reveals two patient clusters, differing by gut-lung interactions and with contrasting clinical phenotypes. A high gut-lung interaction cluster, characterized by lung Pseudomonas, gut Bacteroides, and gut Saccharomyces, is associated with increased exacerbations and greater radiological and overall bronchiectasis severity, whereas the low gut-lung interaction cluster demonstrates an overrepresentation of lung commensals, including Prevotella, Fusobacterium, and Porphyromonas with gut Candida. The lung Pseudomonas-gut Bacteroides relationship, observed in the high gut-lung interaction bronchiectasis cluster, was validated in a murine model of lung Pseudomonas aeruginosa infection. This interaction was abrogated after antibiotic (imipenem) pretreatment in mice confirming the relevance and therapeutic potential of targeting the gut microbiome to influence the gut-lung axis. Metagenomics in a subset of individuals with bronchiectasis corroborated our findings from targeted analyses. Conclusions: A dysregulated gut-lung axis, driven by lung Pseudomonas, associates with poorer clinical outcomes in bronchiectasis.

Squid Suckerin-Spider Silk Fusion Protein Hydrogel for Delivery of Mesenchymal Stem Cell Secretome to Chronic Wounds.

Chronic wounds are non-healing wounds characterized by a prolonged inflammation phase. Excessive inflammation leads to elevated protease levels and consequently to a decrease in growth factors at wound sites. Stem cell secretome therapy has been identified as a treatment strategy to modulate the microenvironment of chronic wounds via supplementation with anti-inflammatory/growth factors. However, there is a need to develop better secretome delivery systems that are able to encapsulate the secretome without denaturation, in a sustained manner, and that are fully biocompatible. To address this gap, a recombinant squid suckerin-spider silk fusion protein is developed with cell-adhesion motifs capable of thermal gelation at physiological temperatures to form hydrogels for encapsulation and subsequent release of the stem cell secretome. Freeze-thaw treatment of the protein hydrogel results in a modified porous cryogel that maintains slow degradation and sustained secretome release. Chronic wounds of diabetic mice treated with the secretome-laden cryogel display increased wound closure, presence of endothelial cells, granulation wound tissue thickness, and reduced inflammation with no fibrotic scar formation. Overall, these in vivo indicators of wound healing demonstrate that the fusion protein hydrogel displays remarkable potential as a delivery system for secretome-assisted chronic wound healing.

Scar prevention through topical delivery of gelatin-tyramine-siSPARC nanoplex loaded in dissolvable hyaluronic acid microneedle patch across skin barrier.

Currently, there is no effective method to prevent the formation of hypertrophic scars and keloids, which can cause severe physical and psychological burdens to patients. Secreted protein acidic and cysteine-rich (SPARC) is involved in wound fibrosis by modulating fibroblast functions, causing excessive collagen deposition during wound healing. Thus, the reduction in SPARC gene expression after wounding can contribute to the downstream reduction in collagen production at the wound site and prevent scar formation. In this study, a dissolvable and biocompatible hyaluronic acid (HA) microneedle patch loaded with nanoplexes containing tyramine-modified gelatin and siRNA for SPARC (siSPARC/Gtn-Tyr) was investigated for topical scar prevention. Tyramine-modified gelatin (Gtn-Tyr) provides electrostatic protection and enhances cell internalization for siSPARC. In vitro studies using human dermal fibroblasts showed that both siSPARC/Gtn-Tyr nanoplexes and siSPARC/Gtn-Tyr-loaded microneedle patches can significantly reduce SPARC gene expression (P < 0.05) and do not cause discernable cytotoxic effects. Further studies using a mouse wound model demonstrate that the siSPARC/Gtn-Tyr-loaded microneedle patch can reduce collagen production during wound healing without triggering an immune response. When Gtn-Tyr-siSPARC is administered transdermally at the wound site, effective collagen reduction is achieved through silencing of the matricellular SPARC protein, thus promising the reduction of scar formation. Overall, the siSPARC/Gtn-Tyr loaded microneedle patch can potentially provide an effective transdermal anti-fibrotic treatment.

Single-cell analysis of skin immune cells reveals an Angptl4-ifi20b axis that regulates monocyte differentiation during wound healing.

The persistent inflammatory response at the wound site is a cardinal feature of nonhealing wounds. Prolonged neutrophil presence in the wound site due to failed clearance by reduced monocyte-derived macrophages delays the transition from the inflammatory to the proliferative phase of wound healing. Angiopoietin-like 4 protein (Angptl4) is a matricellular protein that has been implicated in many inflammatory diseases. However, its precise role in the immune cell response during wound healing remains unclear. Therefore, we performed flow cytometry and single-cell RNA sequencing to examine the immune cell landscape of excisional wounds from Angptl4+/+ and Angptl4-/- mice. Chemotactic immune cell recruitment and infiltration were not compromised due to Angptl4 deficiency. However, as wound healing progresses, Angptl4-/- wounds have a prolonged neutrophil presence and fewer monocyte-derived macrophages than Angptl4+/+ and Angptl4LysM-/- wounds. The underlying mechanism involves a novel Angptl4-interferon activated gene 202B (ifi202b) axis that regulates monocyte differentiation to macrophages, coordinating neutrophil removal and inflammation resolution. An unbiased kinase inhibitor screen revealed an Angptl4-mediated kinome signaling network involving S6K, JAK, and CDK, among others, that modulates the expression of ifi202b. Silencing ifi202b in Angptl4-/- monocytes, whose endogenous expression was elevated, rescued the impaired monocyte-to-macrophage transition in the in vitro reconstituted wound microenvironment using wound exudate. GSEA and IPA functional analyses revealed that ifi202b-associated canonical pathways and functions involved in the inflammatory response and monocyte cell fate were enriched. Together, we identified ifi202b as a key gatekeeper of monocyte differentiation. By modulating ifi202b expression, Angptl4 orchestrates the inflammatory state, innate immune landscape, and wound healing process.

Bioinspired short peptide hydrogel for versatile encapsulation and controlled release of growth factor therapeutics.

A short bioinspired octapeptide, GV8, can self-assemble under mild conditions into biodegradable supramolecular physical hydrogels with high storage modulus and good biocompatibility. GV8 hydrogels can encapsulate both single or multiple macromolecular protein-based therapeutics in a simple one-pot formulation manner, making it a promising candidate to address challenges faced by existing synthetic polymer or peptide hydrogels with complex gelation and drug-encapsulation processes. Alongside its versatility, the hydrogel exhibits concentration-dependent storage modulus and controlled drug-release action. We demonstrate that GV8 hydrogels loaded with adipose-derived mesenchymal stem cells (ADMSC) secretome remain mechanically robust, and exhibit promising potential for wound healing applications by preserving secretome activity while maintaining a constant supply of ADMSC secretome to promote epithelial cell migration. Overall, our work highlights the potential of GV8 peptide hydrogel as a versatile and safe carrier for encapsulation and delivery of macromolecular therapeutics. STATEMENT OF SIGNIFICANCE: Supramolecular peptide hydrogels are a popular choice for protein-based macromolecular therapeutics delivery; however, despite the development of abundant hydrogel systems, several challenges limit their adaptability and practical applications. GV8 short peptide hydrogel circumvents these drawbacks and demonstrates the ability to function as a versatile growth factor (GF) encapsulant. It can encapsulate precise concentrations of complex adipose-derived mesenchymal stem cells secretome mixtures with a one-pot formulation approach and perform controlled release of GFs with preserved activity without compromising the self-assembly and mechanical properties of the hydrogel's supramolecular network. The significance of GV8 hydrogel lies in its gelation simplicity and versatility to encapsulate and deliver macromolecular therapeutics, thus representing a promising biomaterial for regenerative medicine applications.