Proprotein convertase subtilisin/kexin 9 (PCSK9) is responsible for the degradation of the hepatic low-density lipoprotein receptor (LDLR), which regulates circulating cholesterol levels. Consequently, the PCSK9 inhibition is a valuable therapeutic approach for the treatment of hypercholesterolemia and cardiovascular diseases. In our studies, we discovered Rim13, a polyimidazole derivative reducing the protein-protein interaction between PCSK9 and LDLR with an IC50 of 1.6 μM. The computational design led to the optimization of the shape of the PCSK9/ligand complementarity, enabling the discovery of potent diimidazole derivatives. In fact, carrying out biological assays to fully characterize the cholesterol-lowering activity of the new analogues and using both biochemical and cellular techniques, compound Dim16 displayed improved PCSK9 inhibitory activity (IC50 0.9 nM). Interestingly, similar to other lupin-derived peptides and their synthetic analogues, some compounds in this series showed dual hypocholesterolemic activity since some of them complementarily inhibited the 3-hydroxy-3-methylglutaryl coenzyme A reductase.
The term pulmonary hypertension (PH) refers to different conditions, all characterized by increased pressure and resistance in the pulmonary arterial bed. PH has a wide range of causes (essentially, cardiovascular, pulmonary, or connective tissue disorders); however, idiopathic (i.e., without a clear cause) PH exists. This chronic, progressive, and sometimes devastating disease can finally lead to right heart failure and eventually death, through pulmonary vascular remodeling and dysfunction. The exact nature of PH pathophysiology is sometimes still unclear. Extracellular vesicles (EVs), previously known as apoptotic bodies, microvesicles, and exosomes, are small membrane-bound vesicles that are generated by almost all cell types and can be detected in a variety of physiological fluids. EVs are involved in intercellular communication, thus influencing immunological response, inflammation, embryogenesis, aging, and regenerative processes. Indeed, they transport chemokines, cytokines, lipids, RNA and miRNA, and other biologically active molecules. Although the precise functions of EVs are still not fully known, there is mounting evidence that they can play a significant role in the pathophysiology of PH. In this review, after briefly recapping the key stages of PH pathogenesis, we discuss the current evidence on the functions of EVs both as PH biomarkers and potential participants in the distinct pathways of disease progression.
ADP-induced platelet activation leads to cell surface expression of several proteins, including TF (tissue factor). The role of ADP receptors in platelet TF modulation is still unknown. We aimed to assess the (1) involvement of P2Y1 and P2Y12 receptors in ADP-induced TF exposure; (2) modulation of TFpos-platelets in anti-P2Y12-treated patients with coronary artery disease. Based on the obtained results, we revisited the intracellular localization of TF in platelets.The effects of P2Y1 or P2Y12 antagonists on ADP-induced TF expression and activity were analyzed in vitro by flow cytometry and thrombin generation assay in blood from healthy subjects, P2Y12-/-, and patients with gray platelet syndrome. Ex vivo, P2Y12 inhibition of TF expression by clopidogrel/prasugrel/ticagrelor, assessed by VASP (vasodilator-stimulated phosphoprotein) platelet reactivity index, was investigated in coronary artery disease (n=238). Inhibition of open canalicular system externalization and electron microscopy (TEM) were used for TF localization.In blood from healthy subjects, stimulated in vitro by ADP, the percentage of TFpos-platelets (17.3±5.5%) was significantly reduced in a concentration-dependent manner by P2Y12 inhibition only (-81.7±9.5% with 100 nM AR-C69931MX). In coronary artery disease, inhibition of P2Y12 is paralleled by reduction of ADP-induced platelet TF expression (VASP platelet reactivity index: 17.9±11%, 20.9±11.3%, 40.3±13%; TFpos-platelets: 10.5±4.8%, 9.8±5.9%, 13.6±6.3%, in prasugrel/ticagrelor/clopidogrel-treated patients, respectively). Despite this, 15% of clopidogrel good responders had a level of TFpos-platelets similar to the poor-responder group. Indeed, a stronger P2Y12 inhibition (130-fold) is required to inhibit TF than VASP. Thus, a VASP platelet reactivity index <20% (as in prasugrel/ticagrelor-treated patients) identifies patients with TFpos-platelets <20% (92% sensitivity). Finally, colchicine impaired in vitro ADP-induced TF expression but not α-granule release, suggesting that TF is open canalicular system stored as confirmed by TEM and platelet analysis of patients with gray platelet syndrome.Data show that TF expression is regulated by P2Y12 and not P2Y1; P2Y12 antagonists downregulate the percentage of TFpos-platelets. In clopidogrel good-responder patients, assessment of TFpos-platelets highlights those with residual platelet reactivity. TF is stored in open canalicular system, and its membrane exposure upon activation is prevented by colchicine.
Extracellular vesicles (EVs) play a pivotal role in a variety of physiologically relevant processes, including lung inflammation. Recent attention has been directed toward EV-derived microRNAs (miRNAs), such as miR-191-5p, particularly in the context of inflammation. Here, we investigated the impact of miR-191-5p-enriched EVs on the activation of NF-κB and the expression of molecules associated with inflammation such as interleukin-8 (IL-8). To this aim, cells of bronchial epithelial origin, 16HBE, were transfected with miR-191-5p mimic and inhibitor and subsequently subjected to stimulations to generate EVs. Then, bronchial epithelial cells were exposed to the obtained EVs to evaluate the activation of NF-κB and IL-8 levels. Additionally, we conducted a preliminary investigation to analyze the expression profiles of miR-191-5p in EVs isolated from the plasma of patients diagnosed with chronic obstructive pulmonary disease (COPD). Our initial findings revealed two significant observations. First, the exposure of bronchial epithelial cells to miR-191-5p-enriched EVs activated the NF-kB signaling and increased the synthesis of IL-8. Second, we discovered the presence of miR-191-5p in peripheral blood-derived EVs from COPD patients and noted a correlation between miR-191-5p levels and inflammatory and functional parameters. Collectively, these data corroborate and further expand the proinflammatory role of EVs, with a specific emphasis on miR-191-5p as a key cargo involved in this process. Consequently, we propose a model in which miR-191-5p, carried by EVs, plays a role in airway inflammation and may contribute to the pathogenesis of COPD.
Idiopathic pulmonary fibrosis (IPF) is associated with a significantly increased risk of thrombotic events and mortality. This review explores the complex bidirectional relationship between pulmonary fibrosis and thrombosis, discussing epidemiological evidence, pathogenetic mechanisms, and therapeutic implications, with a particular focus on the emerging role of extracellular vesicles (EVs) as crucial mediators linking fibrosis and coagulation. Coagulation factors directly promote fibrosis, while fibrosis itself activates thrombotic pathways. Retrospective studies suggest the benefits of anticoagulants in IPF, but prospective trials have faced challenges. Novel anticoagulants, profibrinolytic therapies, and agents targeting protease-activated receptors (PARs) show promise in preclinical studies and early clinical trials. EVs have emerged as key players in the pathogenesis of interstitial lung diseases (ILDs), serving as vehicles for intercellular communication and contributing to both fibrosis and coagulation. EV-based approaches, such as EV modulation, engineered EVs as drug delivery vehicles, and mesenchymal stem cell-derived EVs, represent promising therapeutic strategies. Ongoing research should focus on optimizing risk–benefit profiles, identifying predictive biomarkers, evaluating combination strategies targeting thrombotic, fibrotic, and inflammatory pathways, and advancing the understanding of EVs in ILDs to develop targeted interventions.
The relative contribution of small (sEVs) and large extracellular vesicles (lEVs) to the total plasma procoagulant potential is not yet well defined. Thus, we compared total and TF
Asthma is the most common chronic respiratory disorder worldwide and accounts for a huge health and economic burden. Its incidence is rapidly increasing but, in parallel, novel personalized approaches have emerged. Indeed, the improved knowledge of cells and molecules mediating asthma pathogenesis has led to the development of targeted therapies that significantly increased our ability to treat asthma patients, especially in severe stages of disease. In such complex scenarios, extracellular vesicles (EVs i.e., anucleated particles transporting nucleic acids, cytokines, and lipids) have gained the spotlight, being considered key sensors and mediators of the mechanisms controlling cell-to-cell interplay. We will herein first revise the existing evidence, mainly by mechanistic studies in vitro and in animal models, that EV content and release is strongly influenced by the specific triggers of asthma. Current studies indicate that EVs are released by potentially all cell subtypes in the asthmatic airways, particularly by bronchial epithelial cells (with different cargoes in the apical and basolateral side) and inflammatory cells. Such studies largely suggest a pro-inflammatory and pro-remodelling role of EVs, whereas a minority of reports indicate protective effects, particularly by mesenchymal cells. The co-existence of several confounding factors—including technical pitfalls and host and environmental confounders—is still a major challenge in human studies. Technical standardization in isolating EVs from different body fluids and careful selection of patients will provide the basis for obtaining reliable results and extend their application as effective biomarkers in asthma.
Depending on local cues, macrophages can polarize into classically activated (M1) or alternatively activated (M2) phenotypes. This study investigates the impact of polarized macrophage-derived Extracellular Vesicles (EVs) (M1 and M2) and their cargo of miRNA-19a-3p and miRNA-425-5p on TGF-β production in lung fibroblasts. EVs were isolated from supernatants of M0, M1, and M2 macrophages and quantified using nanoscale flow cytometry prior to fibroblast stimulation. The concentration of TGF-β in fibroblast supernatants was measured using ELISA assays. The expression levels of miRNA-19a-3p and miRNA-425-5p were assessed via TaqMan-qPCR. TGF-β production after stimulation with M0-derived EVs and with M1-derived EVs increased significantly compared to untreated fibroblasts. miRNA-425-5p, but not miRNA-19a-3p, was significantly upregulated in M2-derived EVs compared to M0- and M1-derived EVs. This study demonstrates that EVs derived from both M0 and M1 polarized macrophages induce the production of TGF-β in fibroblasts, with potential regulation by miRNA-425-5p.
Cell-derived extracellular vesicles (EVs) found in the circulation and body fluids contain biomolecules that could be used as biomarkers for lung and other diseases. EVs from bronchoalveolar lavage (BAL) might be more informative of lung abnormalities than EVs from blood, where information might be diluted. To compare EVs’ characteristics in BAL and blood in smokers with and without COPD. Same-day BAL and blood samples were obtained in 9 nonsmokers (NS), 11 smokers w/o COPD (S), and 9 with COPD (SCOPD) (FEV1: 59 ± 3% pred). After differential centrifugation, EVs (200–500 nm diameter) were identified by flow cytometry and labeled with cell-type specific antigens: CD14 for macrophage-derived EVs, CD326 for epithelial-derived EVs, CD146 for endothelial-derived EVs, and CD62E for activated-endothelial-derived EVs. In BAL, CD14-EVs were increased in S compared to NS [384 (56–567) vs. 172 (115–282) events/μL; p = 0.007] and further increased in SCOPD [619 (224–888)] compared to both S (p = 0.04) and NS (p < 0.001). CD326-EVs were increased in S [760 (48–2856) events/μL, p < 0.001] and in SCOPD [1055 (194–11,491), p < 0.001] when compared to NS [15 (0–68)]. CD146-EVs and CD62E-EVs were similar in the three groups. In BAL, significant differences in macrophage and epithelial-derived EVs can be clearly detected between NS, S and SCOPD, while these differences were not found in plasma. This suggests that BAL is a better medium than blood to study EVs in lung diseases.
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