Exploring the role of RNASET2 in the immune response of black soldier fly larvae.

T2 RNases are transferase-type enzymes distributed across phyla, crucial for breaking down single-stranded RNA molecules. In addition to their canonical function, several T2 enzymes exhibit pleiotropic roles, contributing to various biological processes, such as the immune response in invertebrates and vertebrates. This study aims at characterizing RNASET2 in the larvae of black soldier fly (BSF), Hermetia illucens, which are used for organic waste reduction and the production of valuable insect biomolecules for feed formulation and other applications. Given the exposure of BSF larvae to pathogens present in the feeding substrate, it is likely that the mechanisms of their immune response have undergone significant evolution and increased complexity. After in silico characterization of HiRNASET2, demonstrating the high conservation of this T2 homolog, we investigated the expression pattern of the enzyme in the fat body and hemocytes, two districts mainly involved in the insect immune response, in larvae challenged with bacterial infection. While no variation in HiRNASET2 expression was observed in the fat body following infection, a significant upregulation of HiRNASET2 synthesis occurred in hemocytes shortly after the injection of bacteria in the larva. The intracellular localization of HiRNASET2 in lysosomes of plasmatocytes, its extracellular association with bacteria, and the presence of a putative antimicrobial domain in the molecule, suggest its potential role in RNA clean-up and as an alarm molecule promoting phagocytosis activation by hemocytes. These insights contribute to the characterization of the immune response of Hermetia illucens larvae and may facilitate the development of animal feedstuff enriched with highly valuable BSF bioactive compounds.

Dynamic relationship among extracellular matrix and body wall cells in Hirudo verbana morphogenesis.

A great bulk of recent experimental evidence suggests the key role of the complex crosstalk between the extracellular matrix (ECM) and the cellular component of tissues during morphogenesis and embryogenesis. In particular, remodeling of the ECM and of its physical interactions pattern with surrounding cells represent two crucial processes that might be involved in muscle development. However, little information is available on this topic, especially on invertebrate species. To obtain new insights on how tuning the ECM microenvironment might drive cellular fate during embryonic development, we used the invertebrate medicinal leech Hirudo verbana as a valuable experimental model, due to its simple anatomy and the recapitulation of many aspects of the basic biological processes of vertebrates. Our previous studies on leech post-embryonic development have already shown the pivotal role of ECM changes during the growth of the body wall and the role of Yes-associated protein 1 (YAP1) in mechanotransduction. Here, we suggest that the interactions between stromal cell telocytes and ECM might be crucial in driving the organization of muscle layers during embryogenesis. Furthermore, we propose a possible role of the pleiotropic enzyme HvRNASET2 as a possible modulator of collagen deposition and ECM remodeling not only during regenerative processes (as previously demonstrated) but also in embryogenesis.

A broad-taxa approach as an important concept in ecotoxicological studies and pollution monitoring.

Aquatic invertebrates play a pivotal role in (eco)toxicological assessments because they offer ethical, cost-effective and repeatable testing options. Additionally, their significance in the food chain and their ability to represent diverse aquatic ecosystems make them valuable subjects for (eco)toxicological studies. To ensure consistency and comparability across studies, international (eco)toxicology guidelines have been used to establish standardised methods and protocols for data collection, analysis and interpretation. However, the current standardised protocols primarily focus on a limited number of aquatic invertebrate species, mainly from Arthropoda, Mollusca and Annelida. These protocols are suitable for basic toxicity screening, effectively assessing the immediate and severe effects of toxic substances on organisms. For more comprehensive and ecologically relevant assessments, particularly those addressing long-term effects and ecosystem-wide impacts, we recommended the use of a broader diversity of species, since the present choice of taxa exacerbates the limited scope of basic ecotoxicological studies. This review provides a comprehensive overview of (eco)toxicological studies, focusing on major aquatic invertebrate taxa and how they are used to assess the impact of chemicals in diverse aquatic environments. The present work supports the use of a broad-taxa approach in basic environmental assessments, as it better represents the natural populations inhabiting various ecosystems. Advances in omics and other biochemical and computational techniques make the broad-taxa approach more feasible, enabling mechanistic studies on non-model organisms. By combining these approaches with in vitro techniques together with the broad-taxa approach, researchers can gain insights into less-explored impacts of pollution, such as changes in population diversity, the development of tolerance and transgenerational inheritance of pollution responses, the impact on organism phenotypic plasticity, biological invasion outcomes, social behaviour changes, metabolome changes, regeneration phenomena, disease susceptibility and tissue pathologies. This review also emphasises the need for harmonised data-reporting standards and minimum annotation checklists to ensure that research results are findable, accessible, interoperable and reusable (FAIR), maximising the use and reusability of data. The ultimate goal is to encourage integrated and holistic problem-focused collaboration between diverse scientific disciplines, international standardisation organisations and decision-making bodies, with a focus on transdisciplinary knowledge co-production for the One-Health approach.

Step-by-Step Regeneration of Tentacles after Injury in Anemonia viridis-Morphological and Structural Cell Analyses.

Benthic marine invertebrates, such as corals, are often subjected to injury caused by several sources. Here, the differences and characteristics in injured and health tissues in terms of cellular components are shown through a histological investigation of the soft coral Anemonia viridis at 0 h, 6 h, 24 h, and 7 days after injury caused by tentacle amputation. In addition, a new tool was used for the first time in invertebrates, positron emission tomography, in order to investigate the events that occur during regeneration within a longer time period (0 h, 24 h, and 14 days after the tentacles were cut). Higher integrated density values were measured through a densitometric analysis in sections stained with Fontana-Masson at 24 h after the tentacles were cut. This suggests an increase in melanin-like containing cells and a subsequent increase in fibroblast-like cells differentiated by amoebocytes that converge to the lesion site in the early stages of inflammation and regeneration. This work provides, for the first time, an elucidation of the events that occur during wound-healing and regeneration in basal metazoan, focusing on the characterisation of immune cells and their role. Our results indicate that Mediterranean anthozoan proves to be a valuable model for studying regeneration. Many events highlighted in this research occur in different phyla, suggesting that they are highly conserved.

TERT Extra-Telomeric Roles: Antioxidant Activity and Mitochondrial Protection.

Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase holoenzyme, which adds telomeric DNA repeats on chromosome ends to counteract telomere shortening. In addition, there is evidence of TERT non-canonical functions, among which is an antioxidant role. In order to better investigate this role, we tested the response to X-rays and H2O2 treatment in hTERT-overexpressing human fibroblasts (HF-TERT). We observed in HF-TERT a reduced induction of reactive oxygen species and an increased expression of the proteins involved in the antioxidant defense. Therefore, we also tested a possible role of TERT inside mitochondria. We confirmed TERT mitochondrial localization, which increases after oxidative stress (OS) induced by H2O2 treatment. We next evaluated some mitochondrial markers. The basal mitochondria quantity appeared reduced in HF-TERT compared to normal fibroblasts and an additional reduction was observed after OS; nevertheless, the mitochondrial membrane potential and morphology were better conserved in HF-TERT. Our results suggest a protective function of TERT against OS, also preserving mitochondrial functionality.

Spatio-Temporal Changes of Extracellular Matrix (ECM) Stiffness in the Development of the Leech Hirudo verbana.

The invertebrate leech Hirudo verbana represents a powerful experimental animal model for improving the knowledge about the functional interaction between the extracellular matrix (ECM) and cells within the tissue microenvironment (TME), and the key role played by ECM stiffness during development and growth. Indeed, the medicinal leech is characterized by a simple anatomical organization reproducing many aspects of the basic biological processes of vertebrates and in which a rapid spatiotemporal development is well established and easily assessed. Our results show that ECM structural organization, as well as the amount of fibrillar and non-fibrillar collagen are deeply different from hatching leeches to adult ones. In addition, the changes in ECM remodelling occurring during the different leech developmental stages, leads to a gradient of stiffness regulating both the path of migratory cells and their fates. The ability of cells to perceive and respond to changes in ECM composition and mechanics strictly depend on nuclear or cytoplasmic expression of Yes-Associated Protein 1 (YAP1), a key mediator converting mechanical signals into transcriptional outputs, expression, and activation.

Hirudo verbana as a freshwater invertebrate model to assess the effects of polypropylene micro and nanoplastics dispersion in freshwater.

Plastics are a heterogeneous class of synthetic compounds that, due to their unique characteristics find numerous applications both in industrial and civil fields. However, despite the great advantages that these materials brought in everyday life, the plastic wastes resulting from their massive use represent one of the main environmental problems at the global level. Once released, plastics persist for a long time and are subjected both to biotic and abiotic processes leading to the formation of small particles, known as micro and to nanoplastics, that interact with organisms, accumulating inside tissues and risking to enter in the trophic chain. Among the different types of plastic, polypropylene (PP) is one of the diffused, widely exploited in food and textile industries for disposable packaging and to produce surgical masks. Owing to the huge distribution and the resultant abundant presence of PP waste products, it results necessary investigate the possible toxicity on living organisms. For these reasons, here we analyzed the effects of PP micro and nanoplastics dispersed in freshwater, using the medicinal leech Hirudo verbana as invertebrate model. To better follow the plastics fate, fluorescent particles, labeled with a fluorophore, have been used. Animals were examined at various timings after plastics exposure and results were analyzed by means of microscopy, immunofluorescent and molecular biology analyses. After assessing the entrance of PP fragments into leech tissues, the activation of the innate immune response was evaluated. The results show that the presence of micro and nanoplastics induces an initial physical protection that consists in the secretion of mucus, followed by an increase of blood vessels and the recruitment of immune cells, in particular macrophages. Moreover, macrophages were directly involved in both phagocytic and encapsulation processes, as demonstrated by acid phosphatase (ACP) histoenzymatic and Thioflavin-T assays, expressing specific pro-inflammatory factors, such as HvRNASET2 and HmAIF-1, as demonstrated by immunolocalization and qPCR experiments. Finally, the expression levels of genes related to oxidative stress-induced enzymes have been investigated, in order to evaluate the possible increase in reactive oxygen species (ROS), due to the entry into the leech tissues of PP micro and nanoplastics. This work allows deepening the current knowledge of the possible harmful effects on human health deriving from micro and nanoplastics dispersion, leading new insight about freshwater ecosystems that often represent the first environments interested in plastic pollution.

Amyloid fil rouge from invertebrate up to human ageing: A focus on Alzheimer disease.

Amyloid fibrils and fibril-like structures are currently estimated to represent many different products of several genes in humans and play a key role in many types of proteinopathies, commonly associated with ageing process. They share the mutual feature of aggregation-prone proteins and the building up of molecular-supramolecular structure, such as inter-neuronal plaques in the brain of Alzheimer's Disease (AD) patients, characterized by an extraordinary strength. Noteworthy, this type of structure has been reported in different organisms, in particular in invertebrates. The aim of the current review is to focus on alpha and beta amyloids i.e., SAAs, SAP and APP, elucidating the structure and function of amyloid proteins in invertebrates (such as nematods, annelids, molluscs, insects, ascidians) and highlighting their striking pattern of functional conservation when compared to human amyloid-like fibrils, thus focusing on possible new studies and applications for innovative therapies, particularly for AD, the most common and worldwide type of dementia.

Invertebrate Models in Innate Immunity and Tissue Remodeling Research.

The aim of this Special Issue is to highlight the close functional and highly conserved link between innate immunity, homeostasis maintenance, inflammation, tissue remodeling and regeneration [...].

Identification and Functional Characterization of Toxoneuron nigriceps Ovarian Proteins Involved in the Early Suppression of Host Immune Response.

The endophagous parasitoid Toxoneuron nigriceps (Viereck) (Hymenoptera, Braconidae) of the larval stages of the tobacco budworm Heliothis virescens (Fabricius) (Lepidoptera, Noctuidae) injects the egg, the venom, the calyx fluid, which includes a Polydnavirus (T. nigriceps BracoVirus: TnBV) and the Ovarian Proteins (OPs) into the host body during oviposition. The host metabolism and immune system are disrupted prematurely shortly after parasitization by the combined action of the TnBV, venom, and OPs. OPs are involved in the early suppression of host immune response, before TnBV infects and expresses its genes in the host tissues. In this work, we evaluated the effect of HPLC fractions deriving from in toto OPs. Two fractions caused a reduction in hemocyte viability and were subsequently tested to detect changes in hemocyte morphology and functionality. The two fractions provoked severe oxidative stress and actin cytoskeleton disruption, which might explain the high rate of hemocyte mortality, loss of hemocyte functioning, and hence the host's reduced hemocyte encapsulation ability. Moreover, through a transcriptome and proteomic approach we identify the proteins of the two fractions: eight proteins were identified that might be involved in the observed host hemocyte changes. Our findings will contribute to a better understanding of the secreted ovarian components and their role in parasitoid wasp strategy for evading host immune responses.

Pectin-based bioinks for 3D models of neural tissue produced by a pH-controlled kinetics.

Introduction: In the view of 3D-bioprinting with cell models representative of neural cells, we produced inks to mimic the basic viscoelastic properties of brain tissue. Moving from the concept that rheology provides useful information to predict ink printability, this study improves and expands the potential of the previously published 3D-reactive printing approach by introducing pH as a key parameter to be controlled, together with printing time. Methods: The viscoelastic properties, printability, and microstructure of pectin gels crosslinked with CaCO3 were investigated and their composition was optimized (i.e., by including cell culture medium, HEPES buffer, and collagen). Different cell models representative of the major brain cell populations (i.e., neurons, astrocytes, microglial cells, and oligodendrocytes) were considered. Results and Discussion: The outcomes of this study propose a highly controllable method to optimize the printability of internally crosslinked polysaccharides, without the need for additives or post-printing treatments. By introducing pH as a further parameter to be controlled, it is possible to have multiple (pH-dependent) crosslinking kinetics, without varying hydrogel composition. In addition, the results indicate that not only cells survive and proliferate following 3D-bioprinting, but they can also interact and reorganize hydrogel microstructure. Taken together, the results suggest that pectin-based hydrogels could be successfully applied for neural cell culture.

Insights Into the Immune Response of the Black Soldier Fly Larvae to Bacteria.

In insects, a complex and effective immune system that can be rapidly activated by a plethora of stimuli has evolved. Although the main cellular and humoral mechanisms and their activation pathways are highly conserved across insects, the timing and the efficacy of triggered immune responses can differ among different species. In this scenario, an insect deserving particular attention is the black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae). Indeed, BSF larvae can be reared on a wide range of decaying organic substrates and, thanks to their high protein and lipid content, they represent a valuable source of macromolecules useful for different applications (e.g., production of feedstuff, bioplastics, and biodiesel), thus contributing to the development of circular economy supply chains for waste valorization. However, decaying substrates bring the larvae into contact with different potential pathogens that can challenge their health status and growth. Although these life strategies have presumably contributed to shape the evolution of a sophisticated and efficient immune system in this dipteran, knowledge about its functional features is still fragmentary. In the present study, we investigated the processes underpinning the immune response to bacteria in H. illucens larvae and characterized their reaction times. Our data demonstrate that the cellular and humoral responses in this insect show different kinetics: phagocytosis and encapsulation are rapidly triggered after the immune challenge, while the humoral components intervene later. Moreover, although both Gram-positive and Gram-negative bacteria are completely removed from the insect body within a few hours after injection, Gram-positive bacteria persist in the hemolymph longer than do Gram-negative bacteria. Finally, the activity of two key actors of the humoral response, i.e., lysozyme and phenoloxidase, show unusual dynamics as compared to other insects. This study represents the first detailed characterization of the immune response to bacteria of H. illucens larvae, expanding knowledge on the defense mechanisms of this insect among Diptera. This information is a prerequisite to manipulating the larval immune response by nutritional and environmental factors to increase resistance to pathogens and optimize health status during mass rearing.

An antibody-based enzymatic therapy for cancer treatment: The selective localization of D-amino acid oxidase to EDA fibronectin.

In order to generate an antibody directed enzyme prodrug therapy, here we designed a chimeric protein by fusing the F8 antibody that recognizes the EDA of fibronectin (expressed on the tumor neovasculature) and an evolved variant of the ROS-generating enzyme D-amino acid oxidase (DAAO). The F8(scFv)-DAAO-Q144R recombinant protein is expressed by both CHO-S and E. coli cells. The F8(scFv)-DAAO-Q144R from E. coli cells is fully soluble, shows a high specific activity, is more thermostable in blood than the native DAAO, possesses a binding affinity for EDA well suited for efficient tumor accumulation, and localizes in tumor tissues. Notably, the F8(scFv)-DAAO-Q144R conjugate generates a stronger cytotoxicity to tumor cells than the native enzyme, especially when an inhibitor of heme oxygenase-1 (HO-1) is used, making it a promising candidate for a selective antitumor oxidative therapy controlled by the substrate addition, in the so called "activity on demand", thus sparing normal tissue from damage.

Mesoglea Extracellular Matrix Reorganization during Regenerative Process in Anemonia viridis (Forskål, 1775).

Given the anatomical simplicity and the extraordinary ability to regenerate missing parts of the body, Cnidaria represent an excellent model for the study of the mechanisms regulating regenerative processes. They possess the mesoglea, an amorphous and practically acellular extracellular matrix (ECM) located between the epidermis and the gastrodermis of the body and tentacles and consists of the same molecules present in the ECM of vertebrates, such as collagen, laminin, fibronectin and proteoglycans. This feature makes cnidarians anthozoans valid models for understanding the ECM role during regenerative processes. Indeed, it is now clear that its role in animal tissues is not just tissue support, but instead plays a key role during wound healing and tissue regeneration. This study aims to explore regenerative events after tentacle amputation in the Mediterranean anemone Anemonia viridis, focusing in detail on the reorganization of the ECM mesoglea. In this context, both enzymatic, biometric and histological experiments reveal how this gelatinous connective layer plays a fundamental role in the correct restoration of the original structures by modifying its consistency and stiffness. Indeed, through the deposition of collagen I, it might act as a scaffold and as a guide for the reconstruction of missing tissues and parts, such as amputated tentacles.

HvRNASET2 Regulate Connective Tissue and Collagen I Remodeling During Wound Healing Process.

Several studies have recently demonstrated that the correct regeneration of damaged tissues and the maintaining of homeostasis after wounds or injuries are tightly connected to different biological events, involving immune response, fibroplasia, and angiogenetic processes, in both vertebrates and invertebrates. In this context, our previous data demonstrated that the Hirudo verbana recombinant protein rHvRNASET2 not only plays a pivotal role in innate immune modulation, but is also able to activate resident fibroblasts leading to new collagen production, both in vivo and in vitro. Indeed, when injected in the leech body wall, which represents a consolidated invertebrate model for studying both immune response and tissue regeneration, HvRNASET2 induces macrophages recruitment, fibroplasia, and synthesis of new collagen. Based on this evidence, we evaluate the role of HvRNASET2 on muscle tissue regeneration and extracellular matrix (ECM) remodeling in rHvRNASET2-injected wounded leeches, compared to PBS-injected wounded leeches used as control. The results presented here not only confirms our previous evidence, reporting that HvRNASET2 leads to an increased collagen production, but also shows that an overexpression of this protein might influence the correct progress of muscle tissue regeneration. Moreover, due to its inhibitory effect on vasculogenesis and angiogenesis, HvRNASET2 apparently interfere with the recruitment of the myoendothelial vessel-associated precursor cells that in turn are responsible for muscle regeneration during wound healing repair.

Circulating extracellular vesicles release oncogenic miR-424 in experimental models and patients with aggressive prostate cancer.

Extracellular vesicles (EVs) are relevant means for transferring signals across cells and facilitate propagation of oncogenic stimuli promoting disease evolution and metastatic spread in cancer patients. Here, we investigated the release of miR-424 in circulating small EVs or exosomes from prostate cancer patients and assessed the functional implications in multiple experimental models. We found higher frequency of circulating miR-424 positive EVs in patients with metastatic prostate cancer compared to patients with primary tumors and BPH. Release of miR-424 in small EVs was enhanced in cell lines (LNCaPabl), transgenic mice (Pb-Cre4;Ptenflox/flox;Rosa26ERG/ERG) and patient-derived xenograft (PDX) models of aggressive disease. EVs containing miR-424 promoted stem-like traits and tumor-initiating properties in normal prostate epithelial cells while enhanced tumorigenesis in transformed prostate epithelial cells. Intravenous administration of miR-424 positive EVs to mice, mimicking blood circulation, promoted miR-424 transfer and tumor growth in xenograft models. Circulating miR-424 positive EVs from patients with aggressive primary and metastatic tumors induced stem-like features when supplemented to prostate epithelial cells. This study establishes that EVs-mediated transfer of miR-424 across heterogeneous cell populations is an important mechanism of tumor self-sustenance, disease recurrence and progression. These findings might indicate novel approaches for the management and therapy of prostate cancer.

Role of Ovarian Proteins Secreted by Toxoneuron nigriceps (Viereck) (Hymenoptera, Braconidae) in the Early Suppression of Host Immune Response.

Toxoneuron nigriceps (Viereck) (Hymenoptera, Braconidae) is an endophagous parasitoid of the larval stages of the tobacco budworm, Heliothis virescens (Fabricius) (Lepidoptera, Noctuidae). During oviposition, T. nigriceps injects into the host body, along with the egg, the venom, the calyx fluid, which contains a Polydnavirus (T. nigriceps BracoVirus: TnBV), and the Ovarian Proteins (OPs). Although viral gene expression in the host reaches detectable levels after a few hours, a precocious disruption of the host metabolism and immune system is observed right after parasitization. This alteration appears to be induced by female secretions including TnBV venom and OPs. OPs, originating from the ovarian calyx cells, are involved in the induction of precocious symptoms in the host immune system alteration. It is known that OPs in braconid and ichneumonid wasps can interfere with the cellular immune response before Polydnavirus infects and expresses its genes in the host tissues. Here we show that T. nigriceps OPs induce several alterations on host haemocytes that trigger cell death. The OP injection induces an extensive oxidative stress and a disorganization of actin cytoskeleton and these alterations can explain the high-level of haemocyte mortality, the loss of haemocyte functionality, and so the reduction in encapsulation ability by the host.

Human Primary Dermal Fibroblasts Interacting with 3-Dimensional Matrices for Surgical Application Show Specific Growth and Gene Expression Programs.

Several types of 3-dimensional (3D) biological matrices are employed for clinical and surgical applications, but few indications are available to guide surgeons in the choice among these materials. Here we compare the in vitro growth of human primary fibroblasts on different biological matrices commonly used for clinical and surgical applications and the activation of specific molecular pathways over 30 days of growth. Morphological analyses by Scanning Electron Microscopy and proliferation curves showed that fibroblasts have different ability to attach and proliferate on the different biological matrices. They activated similar gene expression programs, reducing the expression of collagen genes and myofibroblast differentiation markers compared to fibroblasts grown in 2D. However, differences among 3D matrices were observed in the expression of specific metalloproteinases and interleukin-6. Indeed, cell proliferation and expression of matrix degrading enzymes occur in the initial steps of interaction between fibroblast and the investigated meshes, whereas collagen and interleukin-6 expression appear to start later. The data reported here highlight features of fibroblasts grown on different 3D biological matrices and warrant further studies to understand how these findings may be used to help the clinicians choose the correct material for specific applications.

TRPV4 and TRPM8 as putative targets for chronic low back pain alleviation.

The purpose of this study is to investigate the presence of nervous fibers and expression of TRP channels in samples harvested during decompressive/fusion spine surgeries from patients affected by chronic low back pain (CLBP). The aim was to understand if members of this family of receptors played a role in detection and processing of painful stimuli, to eventually define them as potential targets for CLBP alleviation. Expression of transient receptor potential (TRP) channels (A1, V1, V2, V4, and M8) was evaluated in samples from different periarticular sites of 6 patients affected by CLBP, at both protein and transcript levels. The capsular connective pathological tissue appeared infiltrated by sensitive unmyelinated nervous fibers. An increase in TRP channel mRNAs and proteins was observed in the pathological capsule compared with tissues collected from the non-symptomatic area in five of the six analyzed patients, independently by the location and number of affected sites. In particular, TRPV4 and TRPM8 were consistently upregulated in pathological tissues. Interestingly, the only patient showing a different pattern of expression also had a different clinical history. TRPV4 and TRPM8 channels may play a role in CLBP and warrant further investigations as possible therapeutic targets.

CD31+ Extracellular Vesicles From Patients With Type 2 Diabetes Shuttle a miRNA Signature Associated With Cardiovascular Complications.

Innovative biomarkers are needed to improve the management of patients with type 2 diabetes mellitus (T2DM). Blood circulating miRNAs have been proposed as a potential tool to detect T2DM complications, but the lack of tissue specificity, among other reasons, has hampered their translation to clinical settings. Extracellular vesicle (EV)-shuttled miRNAs have been proposed as an alternative approach. Here, we adapted an immunomagnetic bead-based method to isolate plasma CD31+ EVs to harvest vesicles deriving from tissues relevant for T2DM complications. Surface marker characterization showed that CD31+ EVs were also positive for a range of markers typical of both platelets and activated endothelial cells. After characterization, we quantified 11 candidate miRNAs associated with vascular performance and shuttled by CD31+ EVs in a large (n = 218) cross-sectional cohort of patients categorized as having T2DM without complications, having T2DM with complications, and control subjects. We found that 10 of the tested miRNAs are affected by T2DM, while the signature composed by miR-146a, -320a, -422a, and -451a efficiently identified T2DM patients with complications. Furthermore, another CD31+ EV-shuttled miRNA signature, i.e., miR-155, -320a, -342-3p, -376, and -422a, detected T2DM patients with a previous major adverse cardiovascular event. Many of these miRNAs significantly correlate with clinical variables held to play a key role in the development of complications. In addition, we show that CD31+ EVs from patients with T2DM are able to promote the expression of selected inflammatory mRNAs, i.e., CCL2, IL-1α, and TNFα, when administered to endothelial cells in vitro. Overall, these data suggest that the miRNA cargo of plasma CD31+ EVs is largely affected by T2DM and related complications, encouraging further research to explore the diagnostic potential and the functional role of these alterations.