Unveiling the Potential of Extracellular Vesicles as Biomarkers and Therapeutic Nanotools for Gastrointestinal Diseases.

Extracellular vesicles (EVs), acting as inherent nanocarriers adept at transporting a range of different biological molecules such as proteins, lipids, and genetic material, exhibit diverse functions within the gastroenteric tract. In states of normal health, they participate in the upkeep of systemic and organ homeostasis. Conversely, in pathological conditions, they significantly contribute to the pathogenesis of gastrointestinal diseases (GIDs). Isolating EVs from patients' biofluids facilitates the discovery of new biomarkers that have the potential to offer a rapid, cost-effective, and non-invasive method for diagnosing and prognosing specific GIDs. Furthermore, EVs demonstrate considerable therapeutic potential as naturally targeted physiological carriers for the intercellular delivery of therapeutic cargo molecules or as nanoscale tools engineered specifically to regulate physio-pathological conditions or disease progression. Their attributes including safety, high permeability, stability, biocompatibility, low immunogenicity, and homing/tropism capabilities contribute to their promising clinical therapeutic applications. This review will delve into various examples of EVs serving as biomarkers or nanocarriers for therapeutic cargo in the context of GIDs, highlighting their clinical potential for both functional and structural gastrointestinal conditions. The versatile and advantageous properties of EVs position them as promising candidates for innovative therapeutic strategies in advancing personalized medicine approaches tailored to the gastroenteric tract, addressing both functional and structural GIDs.

Semaglutide Modulates Extracellular Matrix Production of LX-2 Cells via Exosomes and Improves Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD).

Metabolic dysfunction-associated steatotic liver disease (MASLD) is closely related to some metabolic disorders, such as central obesity and type 2 diabetes (T2D). Glucagon-like peptide 1 receptor agonists (GLP-1RAs), such as semaglutide, may have therapeutic roles in MASLD associated with T2D. This study aims to investigate the molecular mechanisms underlying the effectiveness of semaglutide on MASLD in terms of progression from liver steatosis to fibrosis. We characterized exosomes from ten patients with type 2 diabetes (T2D) before (T0) and after 12 months (T12) of treatment with once-weekly subcutaneous semaglutide. Six of ten patients were considered responders to therapy (R) based on MASLD severity downgrading by at least one class according to a validated ultrasonographic (US) score. Normal hepatocytes (HEPA-RG) and stellate (LX-2) cells were challenged with exosomes from R and NR patients, isolated before and after 12 months of therapy. Exosomes from both R and NR patients isolated at T0 significantly affected LX-2 viability. After 12 months of treatment, only those isolated from R patients restored cell viability, whereas those from NR patients did not. No effects were observed on HEPA-RG cells. Exosomes at T12 from R but not from NR patients significantly decreased the production of α-SMA, a marker of LX-2 activation, a liver stellate cell model, and ph-SMAD2 and CTGF, involved in fibrosis processes. TGF-ß1 was not modulated by the exosomes of R and NR patients. As a downstream effect, Vimentin, Collagen 1A1, and Fibronectin extracellular matrix components were also downregulated, as measured by droplets digital PCR. In conclusion, these results shed light on the potential effectiveness of semaglutide in improving liver fibrosis in MASLD.

The 'speck'-tacular oversight of the NLRP3-pyroptosis pathway on gastrointestinal inflammatory diseases and tumorigenesis.

The NLRP3 inflammasome is an intracellular sensor and an essential component of the innate immune system involved in danger recognition. An important hallmark of inflammasome activation is the formation of a single supramolecular punctum, known as a speck, per cell, which is the site where the pro-inflammatory cytokines IL-1ß and IL-18 are converted into their bioactive form. Speck also provides the platform for gasdermin D protein activation, whose N-terminus domain perforates the plasma membrane, allowing the release of mature cytokines alongside with a highly inflammatory form of cell death, namely pyroptosis. Although controlled NLRP3 inflammasome-pyroptosis pathway activation preserves mucosal immunity homeostasis and contributes to host defense, a prolonged trigger is deleterious and could lead, in genetically predisposed subjects, to the onset of inflammatory bowel disease, including Crohn's disease and ulcerative colitis, as well as to gastrointestinal cancer. Experimental evidence shows that the NLRP3 inflammasome has both protective and pathogenic abilities. In this review we highlight the impact of the NLRP3-pyroptosis axis on the pathophysiology of the gastrointestinal tract at molecular level, focusing on newly discovered features bearing pro- and anti-inflammatory and neoplastic activity, and on targeted therapies tested in preclinical and clinical trials.

The oleic/palmitic acid imbalance in exosomes isolated from NAFLD patients induces necroptosis of liver cells via the elongase-6/RIP-1 pathway.

Excessive toxic lipid accumulation in hepatocytes underlies the development of non-alcoholic fatty liver disease (NAFLD), phenotypically characterized by necrosis and steato-fibrosis, whose molecular mechanism is not yet fully understood. Patients with NAFLD display an imbalanced palmitic (PA) to oleic acid (OA) ratio. Moreover, increasing experimental evidence points out a relevant involvement of the exosomal content in disease progression. Aim of the study was to highlight the PA/OA imbalance within circulating exosomes, the subsequent intracellular alterations, and the impact on NALFD. Liver cells were challenged with exosomes isolated from both healthy subjects and NAFLD patients. The exosomal PA/OA ratio was artificially modified, and biological effects were evaluated. A NAFLD-derived exosomal PA/OA imbalance impacts liver cell cycle and cell viability. OA-modified NAFLD-derived exosomes restored cellular viability and proliferation, whereas the inclusion of PA into healthy subjects-derived exosomes negatively affected cell viability. Moreover, while OA reduced the phosphorylation and activation of the necroptosis marker, Receptor-interacting protein 1 (phospho-RIP-1), PA induced the opposite outcome, alongside increased levels of stress fibers, such as vimentin and fibronectin. Administration of NAFLD-derived exosomes led to increased expression of Elongase 6 (ELOVL6), Stearoyl-CoA desaturase 1 (SCD1), Tumor necrosis factor α (TNF-α), Mixed-lineage-kinase-domain-like-protein (MLKL) and RIP-1 in the hepatocytes, comparable to mRNA levels in the hepatocytes of NAFLD patients reported in the Gene Expression Omnibus (GEO) database. Genetic and pharmacological abrogation of ELOVL6 elicited a reduced expression of downstream molecules TNF-α, phospho-RIP-1, and phospho-MLKL upon administration of NAFLD-derived exosomes. Lastly, mice fed with high-fat diet exhibited higher phospho-RIP-1 than mice fed with control diet. Targeting the Elongase 6-RIP-1 signaling pathway offers a novel therapeutic approach for the treatment of the NALFD-induced exosomal PA/OA imbalance.

Encapsulation of MCC950 in Liposomes Decorated with Anti-Frizzled 1 Improves Drug Bioavailability and Effectiveness in Fatty Liver Disease.

Inflammasome activation plays a crucial role in the progression to more severe stages of non-alcoholic fatty liver disease (NAFLD), representing a promising therapeutic target. MCC950 is a small molecule acting as a potent and specific inhibitor of the canonical and non-canonical activation of the NLRP3 inflammasome, but its short plasmatic half-life limits its use. Herein, we report, for the first time, the encapsulation of MCC950 in poly(ethylene glycol) (PEG) liposomes (LPs) that are specifically functionalized with an antibody against Frizzled 1 (FZD1), a g-coupled protein involved in the WNT pathway and overexpressed on inflammasome-activated macrophages. MCC950, encapsulated into PEG-LP formulations conjugated with an anti-FZD1 antibody, inhibits the NLRP3 inflammasome activation at concentrations 10 times lower than that of the free drug in THP-1 cells. Luminescent carbon dots (CDs) were also co-encapsulated with MCC950 in LPs to obtain optically traceable nanoformulations that have proved the enhanced ability of the targeted LPs to be internalized into THP-1 cells with respect to their nontargeted counterparts. Our results suggest that MCC950 encapsulation into targeted LPs represents a valuable strategy to achieve reformulation of the NLRP3 inhibitor, able to significantly curtail the threshold of MCC950 doses for inhibiting inflammasome activation, thus offering a new therapeutic approach.

Laminin-332 γ2 Monomeric Chain Promotes Adhesion and Migration of Hepatocellular Carcinoma Cells.

Extracellular matrix (ECM) has a well-recognized impact on the progression of solid tumors, including hepatocellular carcinoma (HCC). Laminin 332 (Ln332) is a ECM molecule of epithelial basal lamina, composed of three polypeptide chains (α3, ß3, and γ2), that is usually poorly expressed in the normal liver but is detected at high levels in HCC. This macromolecule was shown to promote the proliferation, epithelial-to-mesenchymal transition (EMT), and drug resistance of HCC cells. The monomeric γ2 chain is up-regulated and localized preferentially at the invasive edge of metastatic intrahepatic HCC nodules, suggesting its potential involvement in the acquisition of invasive properties of HCC cells. HCC cells were tested in in vitro adhesion, scattering, and transwell migration assays in response to fibronectin and the Ln332 and Ln332 γ2 chains, and the activation status of major signaling pathways involved was evaluated. Here, we show that the Ln332 γ2 chain promotes HCC the cell adhesion, migration, and scattering of HCC cells that express the Ln332 receptor α3ß1 integrin, proving to be a causal factor of the EMT program achievement. Moreover, we found that efficient HCC cell adhesion and migration on γ2 require the activation of the small cytosolic GTPase Rac1 and ERKs signaling. These data suggest that the γ2 chain, independently from the full-length Ln332, can contribute to the pro-invasive potential of aggressive HCC cell subpopulations.