Interstitial Fluid Shear Stress Induces the Synthetic Phenotype Switching of VSMCs to Release Pro-calcified Extracellular Vesicles via EGFR-MAPK-KLF5 Pathway.

Phenotypic switching (from contractile to synthetic) of vascular smooth muscle cells (VSMCs) is essential in the progression of atherosclerosis. The damaged endothelium in the atherosclerotic artery exposes VSMCs to increased interstitial fluid shear stress (IFSS). However, the precise mechanisms by which increased IFSS influences VSMCs phenotypic switching are unrevealed. Here, we employed advanced numerical simulations to calculate IFSS values accurately based on parameters acquired from patient samples. We then carefully investigated the phenotypic switching and extracellular vesicles (EVs) secretion of VSMCs under various IFSS conditions. By employing a comprehensive set of approaches, we found that VSMCs exhibited synthetic phenotype upon atherosclerotic IFSS. This synthetic phenotype is the upstream regulator for the enhanced secretion of pro-calcified EVs. Mechanistically, as a mechanotransducer, the epidermal growth factor receptor (EGFR) initiates the flow-based mechanical cues to MAPK signaling pathway, facilitating the nuclear accumulation of the transcription factor krüppel-like factor 5 (KLF5). Furthermore, pharmacological inhibiting either EGFR or MAPK signaling pathway blocks the nuclear accumulation of KLF5 and finally results in the maintenance of contractile VSMCs even under increased IFSS stimulation. Collectively, targeting this signaling pathway holds potential as a novel therapeutic strategy to inhibit VSMCs phenotypic switching and mitigate the progression of atherosclerosis.

Integrating Anion-π+ Interaction and Crowded Conformation to Develop Multifunctional NIR AIEgen for Effective Tumor Theranostics via Hippo-YAP Pathway.

The technology of aggregation-induced emission (AIE) presents a promising avenue for fluorescence imaging-guided photodynamic cancer therapy. However, existing near-infrared AIE photosensitizers (PSs) frequently encounter limitations, including tedious synthesis, poor tumor retention, and a limited understanding of the underlying molecular biology mechanism. Herein, an effective molecular design paradigm of anion-π+ interaction combined with the inherently crowded conformation that could enhance fluorescence efficacy and reactive oxygen species generation was proposed through a concise synthetic method. Mechanistically, upon photosensitization, the Hippo signaling pathway contributes to the death of melanoma cells and promotes the nuclear location of its downstream factor, yes-associated protein, which regulates the transcription and expression of apoptosis-related genes. The finding in this study would trigger the development of high-performance and versatile AIE PSs for precision cancer therapy based on a definite regulatory mechanism.

Redox-active endosomes mediate α5ß1 integrin signaling and promote chondrocyte matrix metalloproteinase production in osteoarthritis.

Mechanical cues sensed by integrins induce cells to produce proteases to remodel the extracellular matrix. Excessive protease production occurs in many degenerative diseases, including osteoarthritis, in which articular cartilage degradation is associated with the genesis of matrix protein fragments that can activate integrins. We investigated the mechanisms by which integrin signals may promote protease production in response to matrix changes in osteoarthritis. Using a fragment of the matrix protein fibronectin (FN) to activate the α5ß1 integrin in primary human chondrocytes, we found that endocytosis of the integrin and FN fragment complex drove the production of the matrix metalloproteinase MMP-13. Activation of α5ß1 by the FN fragment, but not by intact FN, was accompanied by reactive oxygen species (ROS) production initially at the cell surface, then in early endosomes. These ROS-producing endosomes (called redoxosomes) contained the integrin-FN fragment complex, the ROS-producing enzyme NADPH oxidase 2 (NOX2), and SRC, a redox-regulated kinase that promotes MMP-13 production. In contrast, intact FN was endocytosed and trafficked to recycling endosomes without inducing ROS production. Articular cartilage from patients with osteoarthritis showed increased amounts of SRC and the NOX2 complex component p67phox. Furthermore, we observed enhanced localization of SRC and p67phox at early endosomes, suggesting that redoxosomes could transmit and sustain integrin signaling in response to matrix damage. This signaling mechanism not only amplifies the production of matrix-degrading proteases but also establishes a self-perpetuating cycle that contributes to the ongoing degradation of cartilage matrix in osteoarthritis.

Killing tumor-associated bacteria with a liposomal antibiotic generates neoantigens that induce anti-tumor immune responses.

Increasing evidence implicates the tumor microbiota as a factor that can influence cancer progression. In patients with colorectal cancer (CRC), we found that pre-resection antibiotics targeting anaerobic bacteria substantially improved disease-free survival by 25.5%. For mouse studies, we designed an antibiotic silver-tinidazole complex encapsulated in liposomes (LipoAgTNZ) to eliminate tumor-associated bacteria in the primary tumor and liver metastases without causing gut microbiome dysbiosis. Mouse CRC models colonized by tumor-promoting bacteria (Fusobacterium nucleatum spp.) or probiotics (Escherichia coli Nissle spp.) responded to LipoAgTNZ therapy, which enabled more than 70% long-term survival in two F. nucleatum-infected CRC models. The antibiotic treatment generated microbial neoantigens that elicited anti-tumor CD8+ T cells. Heterologous and homologous bacterial epitopes contributed to the immunogenicity, priming T cells to recognize both infected and uninfected tumors. Our strategy targets tumor-associated bacteria to elicit anti-tumoral immunity, paving the way for microbiome-immunotherapy interventions.

Intra-articular delivery of AAV vectors encoding PD-L1 attenuates joint inflammation and tissue damage in a mouse model of rheumatoid arthritis.

Objective: Rheumatoid arthritis (RA) is the most common form of autoimmune inflammatory arthritis. Intra-articular gene delivery to block proinflammatory cytokines has been studied in pre-clinical models and human clinical trials. It has been demonstrated that the level of programmed death-ligand 1 (PD-L1) is associated with rheumatoid arthritis (RA). This study examined the therapeutic role of PD-L1 by intra-articular delivery via adeno-associated virus (AAV) vectors in the mouse collagen-induced arthritis (CIA) model. Methods: Mice were intra-articularly injected with AAV5 vectors encoding human PD-L1 on day 0 and immunized with bovine type II collagen to induce CIA simultaneously. On day 49 post AAV administration, joints were collected for histo-pathological and cytokine analysis. Additionally, the systemic impacts of intra-articular injection of AAV5/PD-L1 vectors were also studied. To study the therapeutic effect of PD-L1, AAV5/PD-L1 vectors were administered into the joints of RA mice on day 21. Results: After administration of AAV5/PD-L1 vectors, strong PD-L1 expression was detected in AAV transduced joints. Joints treated with PD-L1 at the time of arthritis induction exhibited significantly less swelling and improved histopathological scores when compared to untreated joints. Additionally, the infiltration of T cells and macrophages was decreased in joints of CIA mice that received AAV5/PD-L1 vectors (P<0.05). The levels of pro-inflammatory cytokines, including IL-1, IL-6, IL-17 and TNFα, were lower in AAV5/PD-L1 treated than untreated joints (P<0.05). Furthermore, the administration of AAV5/PD-L1 vectors into the joints of CIA mice did not impact serum cytokine levels and the antibody titers to type II collagen. Biodistribution of AAV vectors after intra-articular injection showed undetectable AAV genomes in other tissues except for a low level in the liver. Similar to the results of AAV5/PD-L1 vector administration on day 0, decreased joint swelling and lower histopathological damage were observed in joints treated with AAV5/PD-L1 vectors on day 21. Conclusion: The results from this study demonstrate that local AAV mediated PD-L1 gene delivery into the joints is able to prevent the development and block the progression of arthritis in CIA mice without impacting systemic immune responses. This study provides a novel strategy to effectively treat inflammatory joint diseases using local AAV gene therapy by interference with immune checkpoint pathways.

Increased Wnt/ß-catenin signaling contributes to autophagy inhibition resulting from a dietary magnesium deficiency in injury-induced osteoarthritis.

BACKGROUND: Dietary magnesium deficiency, which is common in modern diet, has been associated with osteoarthritis (OA) susceptibility. Despite this clinical association, no study has addressed if dietary magnesium deficiency accelerates OA development, especially at molecular level. This study aimed to explore aggravating effects of dietary magnesium deficiency on cartilage damage in an injury-induced murine OA model and to determine the underlying mechanism. METHODS: Twelve-week-old C57BL/6J mice subject to injury-induced OA modeling were randomized into different diet groups in which the mice were fed a diet with daily recommended magnesium content (500 mg/kg) or diets with low magnesium content (100 or 300 mg/kg). Articular cartilage damage was evaluated using the OARSI score. To determine molecular mechanisms in vitro, mouse chondrocytes were treated with media of low magnesium conditions at 0.1 and 0.4 mM, compared with normal magnesium condition at 0.7 mM as control. Anabolic and catabolic factors, autophagy markers, ß-catenin, Wnt ligands, and a magnesium channel transient receptor potential cation channel subfamily member 7 (TRPM7) were analyzed by quantitative real-time PCR and immunoblotting. Autolysosomes were detected by DALGreen staining via fluorescence microscopy and autophagosomes were evaluated by transmission electron microscopy. Autophagy markers, ß-catenin, and TRPM7 were assessed in vivo in the mouse cartilage, comparing between dietary magnesium deficiency and normal diet, by immunohistochemistry. RESULTS: Dietary magnesium deficiency aggravated injury-induced cartilage damage, indicated by significant higher OARSI scores. Autophagy markers LC3-II and Beclin-1 were decreased both in low magnesium diet-fed mice and low magnesium-treated chondrocytes. The number of autolysosomes and autophagosomes was also reduced under low magnesium conditions. Moreover, magnesium deficiency induced decreased anabolic and increased catabolic effect of chondrocytes which could be restored by autophagy activator rapamycin. In addition, reduced autophagy under low magnesium conditions is mediated by activated Wnt/ß-catenin signaling. The expression of TRPM7 also decreased in low magnesium diet-fed mice, indicating that downstream changes could be regulated through this channel. CONCLUSIONS: Dietary magnesium deficiency contributes to OA development, which is mediated by reduced autophagy through Wnt/ß-catenin signaling activation. These findings indicated potential benefits of adequate dietary magnesium for OA patients or those individuals at high risk of OA.

Identification of potentially functional circRNAs and prediction of circRNA-miRNA-mRNA regulatory network in periodontitis: Bridging the gap between bioinformatics and clinical needs.

BACKGROUND AND OBJECTIVE: Periodontitis is a multifactorial chronic inflammatory disease that can lead to the irreversible destruction of dental support tissues. As an epigenetic factor, the expression of circRNA is tissue-dependent and disease-dependent. This study aimed to identify novel periodontitis-associated circRNAs and predict relevant circRNA-periodontitis regulatory network by using recently developed bioinformatic tools and integrating sequencing profiling with clinical information for getting a better and more thorough image of periodontitis pathogenesis, from gene to clinic. MATERIAL AND METHODS: High-throughput sequencing and RT-qPCR were conducted to identify differentially expressed circRNAs in gingival tissues from periodontitis patients. The relationship between upregulated circRNAs expression and probing depth (PD) was performed using Spearman's correlation analysis. Bioinformatic analyses including GO analysis, circRNA-disease association prediction, and circRNA-miRNA-mRNA network prediction were performed to clarify potential regulatory functions of identified circRNAs in periodontitis. A receiver-operating characteristic (ROC) curve was established to assess the diagnostic significance of identified circRNAs. RESULTS: High-throughput sequencing identified 70 differentially expressed circRNAs (68 upregulated and 2 downregulated circRNAs) in human periodontitis (fold change >2.0 and p < .05). The top five upregulated circRNAs were validated by RT-qPCR that had strong associations with multiple human diseases, including periodontitis. The upregulation of circRNAs were positively correlated with PD (R = .40-.69, p < .05, moderate). A circRNA-miRNA-mRNA network with the top five upregulated circRNAs, differentially expressed mRNAs, and overlapped predicted miRNAs indicated potential roles of circRNAs in immune response, cell apoptosis, migration, adhesion, and reaction to oxidative stress. The ROC curve showed that circRNAs had potential value in periodontitis diagnosis (AUC = 0.7321-0.8667, p < .05). CONCLUSION: CircRNA-disease associations were predicted by online bioinformatic tools. Positive correlation between upregulated circRNAs, circPTP4A2, chr22:23101560-23135351+, circARHGEF28, circBARD1 and circRASA2, and PD suggested function of circRNAs in periodontitis. Network prediction further focused on downstream targets regulated by circRNAs during periodontitis pathogenesis.

The imbalance between regulatory and memory B cells accompanied by an increased number of circulating T-follicular helper cells in MOG-antibody-associated demyelination.

OBJECTIVE: To explore the alteration of T and B lymphocyte subsets proportions in myelin oligodendrocyte glycoprotein (MOG)-antibody-associated demyelination. METHODS: 19 MOG-antibody-positive, 25 AQP4-antibody-positive and 25 double-negative NMOSD patients in the acute phase of the diseases were included in the study, as well as 29 healthy controls. The frequencies of different lymphocyte subsets, including CD19+CD27+ memory B cells, CD19+CD24hiCD38hi, and CD19+CD5+CD1dhi regulatory B cells, IFN-γexpressing B cells, IL-10 expressing B cells and CD4+CXCR5+ICOS+T-follicular helper cells (TFH) were measured via flow cytometry and compared among the four groups. RESULTS: The frequencies of CD19+CD24hiCD38hi, CD19+CD5+CD1dhi regulatory B cells as well as the IL-10 expressing B cells were significantly lower in the MOG-antibody-associated demyelination compared to the healthy controls, whereas the frequencies of CD19+CD27+ memory B cells were significantly higher in the MOG-antibody-positive group. The frequencies of TFH were significantly higher in the MOG-antibody-positive group as compared to the healthy controls. No significant difference was detected in the above mentioned lymphocytic profile between the MOG-antibody-positive and the AQP4-antibody-positive groups. CONCLUSIONS: The immuno-regulatory functions of B cells were significantly impaired whereas TFH cells were markedly increased in the acute phase of MOG-antibody-associated demyelination. Despite having distinct clinical features, MOG-antibody-associated demyelination shared a similar lymphocytic profile with AQP4-antibody-positive NMOSD in the acute relapse phase.