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Plasma extracellular vesicles (EVs) are lipid membrane vesicles involved in several biological processes including coagulation. Both coagulation and lipid metabolism are strongly associated with cardiovascular events. Lowering very-low- and low-density lipoprotein ((V)LDL) particles via dextran sulphate LDL apheresis also removes coagulation proteins. It remains unknown, however, how coagulation proteins are removed in apheresis. We hypothesize that plasma EVs that contain high levels of coagulation proteins are concomitantly removed with (V)LDL particles by dextran sulphate apheresis. For this, we precipitated (V)LDL particles from human plasma with dextran sulphate and analyzed the abundance of coagulation proteins and EVs in the precipitate. Coagulation pathway proteins, as demonstrated by proteomics and a bead-based immunoassay, were over-represented in the (V)LDL precipitate. In this precipitate, both bilayer EVs and monolayer (V)LDL particles were observed by electron microscopy. Separation of EVs from (V)LDL particles using density gradient centrifugation revealed that almost all coagulation proteins were present in the EVs and not in the (V)LDL particles. These EVs also showed a strong procoagulant activity. Our study suggests that dextran sulphate used in LDL apheresis may remove procoagulant EVs concomitantly with (V)LDL particles, leading to a loss of coagulation proteins from the blood. [ABSTRACT FROM AUTHOR]

[Display omitted] Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, and its advanced form non-alcoholic steatohepatitis (NASH) may progress to cirrhosis and hepatocellular carcinoma. Ceramides have been shown to exacerbate NAFLD development through enhancing insulin resistance, reactive oxygen species production, liver steatosis, lipotoxicity and hepatocyte apoptosis, and eventually causing hepatic inflammation and fibrosis. Emerging evidence indicates that ceramide production in NAFLD is predominantly attributed to activation of the de novo synthesis pathway of ceramides in hepatocytes. More importantly, pharmacological modulation of ceramide de novo synthesis in preclinical studies seems efficacious for the treatment of NAFLD. In this review, we provide an overview of the pathogenic mechanisms of ceramides in NAFLD, discuss recent advances and challenges in pharmacological interventions targeting ceramide de novo synthesis, and propose some research directions in the field. [ABSTRACT FROM AUTHOR]

Von Willebrand disease ( VWD) is a bleeding disorder characterized by reduced plasma von Willebrand factor ( VWF) levels or functionally abnormal VWF. Low VWF plasma levels in VWD patients are the result of mutations in the VWF gene that lead to decreased synthesis, impaired secretion, increased clearance or a combination thereof. However, expression studies of variants located in the A domains of VWF are limited. We therefore characterized the biosynthesis of VWF mutations, located in the VWF A1-A3 domains, that were found in families diagnosed with VWD. Human Embryonic Kidney 293 ( HEK293) cells were transiently transfected with plasmids encoding full-length wild-type VWF or mutant VWF. Six mutations in the A1-A3 domains were expressed. We found that all mutants, except one, showed impaired formation of elongated pseudo-Weibel-Palade bodies ( WPB). In addition, two mutations also showed reduced numbers of pseudo- WPB, even in the heterozygous state, and increased endoplasmic reticulum retention, which is in accordance with the impaired regulated secretion seen in patients. Regulated secretion upon stimulation of transfected cells reproduced the in vivo situation, indicating that HEK293 cells expressing VWF variants found in patients with VWD can be used to properly assess defects in regulated secretion. [ABSTRACT FROM AUTHOR]

Involvement of the Toll-like receptor 4 (TLR4) in maladaptive cardiac remodeling and heart failure (HF) upon pressure overload has been studied extensively, but less is known about the role of TLR2. Interplay and redundancy of TLR4 with TLR2 have been reported in other organs but were not investigated during cardiac dysfunction. We explored whether TLR2 deficiency leads to less adverse cardiac remodeling upon chronic pressure overload and whether TLR2 and TLR4 additively contribute to this. We subjected 35 male C57BL/6J mice (wildtype (WT) or TLR2 knockout (KO)) to sham or transverse aortic constriction (TAC) surgery. After 12 weeks, echocardiography and electrocardiography were performed, and hearts were extracted for molecular and histological analysis. TLR2 deficiency (n = 14) was confirmed in all KO mice by PCR and resulted in less hypertrophy (heart weight to tibia length ratio (HW/TL), smaller cross-sectional cardiomyocyte area and decreased brain natriuretic peptide (BNP) mRNA expression, p < 0.05), increased contractility (QRS and QTc, p < 0.05), and less inflammation (e.g., interleukins 6 and 1β, p < 0.05) after TAC compared to WT animals (n = 11). Even though TLR2 KO TAC animals presented with lower levels of ventricular TLR4 mRNA than WT TAC animals (13.2 ± 0.8 vs. 16.6 ± 0.7 mg/mm, p < 0.01), TLR4 mRNA expression was increased in animals with the largest ventricular mass, highest hypertrophy, and lowest ejection fraction, leading to two distinct groups of TLR2 KO TAC animals with variations in cardiac remodeling. This variation, however, was not seen in WT TAC animals even though heart weight/tibia length correlated with expression of TLR4 in these animals (r = 0.078, p = 0.005). Our data suggest that TLR2 deficiency ameliorates adverse cardiac remodeling and that ventricular TLR2 and TLR4 additively contribute to adverse cardiac remodeling during chronic pressure overload. Therefore, both TLRs may be therapeutic targets to prevent or interfere in the underlying molecular processes. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we investigated the mechanism of PLA2-induced lysosomal destabilization. Through the measurements of lysosomal β-hexosaminidase free activity, their membrane potential, the intra-lysosomal pH and the lysosomal latency loss in hypotonic sucrose medium, we established that PLA2 could increase the lysosomal membrane permeability to both potassium ions and protons. The enzyme could also enhance the organelle osmotic sensitivity. The increases in the lysosomal ion permeability promoted influx of potassium ions into the lysosomes via K+/H+ exchange. The resulted osmotic imbalance across the lysosomal membranes osmotically destabilized the lysosomes. In addition, the enhancement of the lysosomal osmotic sensitivity caused the lysosomes to become more liable to destabilization in the osmotic stress. The results explain how PLA2 destabilized the lysosomes. [Copyright &y& Elsevier]

Food‐derived extracellular vesicles (FEVs) are nanoscale membrane vesicles obtained from dietary materials such as breast milk, plants and probiotics. Distinct from other EVs, FEVs can survive the harsh degrading conditions in the gastrointestinal tract and reach the intestines. This unique feature allows FEVs to be promising prebiotics in health and oral nanomedicine for gut disorders, such as inflammatory bowel disease. Interestingly, therapeutic effects of FEVs have recently also been observed in non‐gastrointestinal diseases. However, the mechanisms remain unclear or even mysterious. It is speculated that orally administered FEVs could enter the bloodstream, reach remote organs, and thus exert therapeutic effects therein. However, emerging evidence suggests that the amount of FEVs reaching organs beyond the gastrointestinal tract is marginal and may be insufficient to account for the significant therapeutic effects achieved regarding diseases involving remote organs such as the liver. Thus, we herein propose that FEVs primarily act locally in the intestine by modulating intestinal microenvironments such as barrier integrity and microbiota, thereby eliciting therapeutic impact remotely on the liver in non‐gastrointestinal diseases via the gut‐liver axis. Likewise, drugs delivered to the gastrointestinal system through FEVs may act via the gut‐liver axis. As the liver is the main metabolic hub, the intestinal microenvironment may be implicated in other metabolic diseases. In fact, many patients with non‐alcoholic fatty liver disease, obesity, diabetes and cardiovascular disease suffer from a leaky gut and dysbiosis. In this review, we provide an overview of the recent progress in FEVs and discuss their biomedical applications as therapeutic agents and drug delivery systems, highlighting the pivotal role of the gut‐liver axis in the mechanisms of action of FEVs for the treatment of gut disorders and metabolic diseases. [ABSTRACT FROM AUTHOR]

Aim: Patients with metabolic dysfunction‐associated steatotic liver disease (MASLD) are at increased risk of incident cardiovascular disease. However, the clinical characteristics and prognostic importance of MASLD in patients presenting with acute myocardial infarction (AMI) have yet to be examined. Methods: This study compared the characteristics and outcomes of patients with and without MASLD presenting with AMI at a tertiary centre in Singapore. MASLD was defined as hepatic steatosis, with at least one of five metabolic criteria. Hepatic steatosis was determined using the Hepatic Steatosis Index. Propensity score matching was performed to adjust for age and sex. The Kaplan‐Meier curve was constructed for long‐term all‐cause mortality. Cox regression analysis was used to investigate independent predictors of long‐term all‐cause mortality. Results: In this study of 4446 patients with AMI, 2223 patients with MASLD were matched with patients without MASLD using propensity scores. The mean follow‐up duration was 3.4 ± 2.4 years. The MASLD group had higher rates of obesity, diabetes and chronic kidney disease than their counterparts. Patients with MASLD had early excess all‐cause mortality (6.8% vs. 3.6%, p <.001) at 30 days, with unfavourable mortality rates sustained in the long‐term (18.3% vs. 14.5%, p =.001) compared with those without MASLD. After adjustment, MASLD remained independently associated with higher long‐term all‐cause mortality (hazard ratio 1.330, 95% confidence interval 1.106‐1.598, p =.002). Conclusion: MASLD embodies a higher burden of metabolic dysfunction and is an independent predictor of long‐term mortality in the AMI population. Its early identification may be beneficial for risk stratification and provide therapeutic targets for secondary preventive strategies in AMI. [ABSTRACT FROM AUTHOR]

Background: Dilated cardiomyopathy (DCM) is a severe, non-ischemic heart disease which ultimately results in heart failure (HF). Decades of research on DCM have revealed diverse aetiologies. Among them, familial DCM is the major form of DCM, with pathogenic variants in LMNA being the second most common form of autosomal dominant DCM. LMNA DCM is a multifactorial and complex disease with no specific treatment thus far. Many studies have demonstrated that perturbing candidates related to various dysregulated pathways ameliorate LMNA DCM. However, it is unknown whether these candidates could serve as potential therapeutic targets especially in long term efficacy. Methods: We evaluated 14 potential candidates including Lmna gene products (Lamin A and Lamin C), key signaling pathways (Tgfβ/Smad, mTor and Fgf/Mapk), calcium handling, proliferation regulators and modifiers of LINC complex function in a cardiac specific Lmna DCM model. Positive candidates for improved cardiac function were further assessed by survival analysis. Suppressive roles and mechanisms of these candidates in ameliorating Lmna DCM were dissected by comparing marker gene expression, Tgfβ signaling pathway activation, fibrosis, inflammation, proliferation and DNA damage. Furthermore, transcriptome profiling compared the differences between Lamin A and Lamin C treatment. Results: Cardiac function was restored by several positive candidates (Smad3, Yy1, Bmp7, Ctgf, aYAP1, Sun1, Lamin A, and Lamin C), which significantly correlated with suppression of HF/fibrosis marker expression and cardiac fibrosis in Lmna DCM. Lamin C or Sun1 shRNA administration achieved consistent, prolonged survival which highly correlated with reduced heart inflammation and DNA damage. Importantly, Lamin A treatment improved but could not reproduce long term survival, and Lamin A administration to healthy hearts itself induced DCM. Mechanistically, we identified this lapse as caused by a dose-dependent toxicity of Lamin A, which was independent from its maturation. Conclusions: In vivo candidate evaluation revealed that supplementation of Lamin C or knockdown of Sun1 significantly suppressed Lmna DCM and achieve prolonged survival. Conversely, Lamin A supplementation did not rescue long term survival and may impart detrimental cardiotoxicity risk. This study highlights a potential of advancing Lamin C and Sun1 as therapeutic targets for the treatment of LMNA DCM. Highlights: After evaluation of 14 potential candidates in a cardiac-specific Lmna DCM model, we demonstrated that Smad3 shRNA, Yy1, combination of Bmp7 and Ctgf (Bmp7-Ctgf shRNA), Yap1, Sun1 shRNA, Lamin A, and Lamin C improved cardiac function. Sun1 shRNA and Lamin C particularly prolonged a long-term survival. We uncovered that inflammation and DNA damage markers were among the top list of highly correlated markers in addition to traditional HF and fibrosis markers, suggesting these additional markers are important to evaluate the candidates for the treatment efficacy of LMNA DCM. The study revealed that treating Lmna DCM with Lamin A did not work as expected and had toxic effects. The toxicity was found to be dose-dependent and not caused by prelamin A processing. [ABSTRACT FROM AUTHOR]

Abstract Dietary modifications can help prevent many cardiovascular disease (CVD) events. Endothelial progenitor cells (EPCs) actively contribute to cardiovascular system maintenance and could function as surrogate markers for evaluating improvement in cardiovascular health resulting from nutritional interventions. This review summarizes the latest research progress on the impact of food and nutrients on EPCs, drawing on evidence from human, animal, and in vitro studies. Additionally, current trends and challenges faced in the field are highlighted. Findings from studies examining cells as EPCs are generally consistent, demonstrating that a healthy diet, such as the Mediterranean diet or a supervised diet for overweight people, specific foods like olive oil, fruit, vegetables, red wine, tea, chia, and nutraceuticals, and certain nutrients such as polyphenols, unsaturated fats, inorganic nitrate, and vitamins, generally promote higher EPC numbers and enhanced EPC function. Conversely, an unhealthy diet, such as one high in sugar substitutes, salt, or fructose, impairs EPC function. Research on outgrowth EPCs has revealed that various pathways are involved in the modulation effects of food and nutrients. The potential of EPCs as a biomarker for assessing the effectiveness of nutritional interventions in preventing CVDs is immense, while further clarification on definition and characterization of EPCs is required. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) can be produced from red blood cells (RBCs) on a large scale and used to deliver therapeutic payloads efficiently. However, not much is known about the native biological properties of RBCEVs. Here, we demonstrate that RBCEVs are primarily taken up by macrophages and monocytes. This uptake is an active process, mediated mainly by endocytosis. Incubation of CD14+ monocytes with RBCEVs induces their differentiation into macrophages with an Mheme‐like phenotype, characterized by upregulation of heme oxygenase‐1 (HO‐1) and the ATP‐binding cassette transporter ABCG1. Moreover, macrophages that take up RBCEVs exhibit a reduction in surface CD86 and decreased secretion of TNF‐α under inflammatory stimulation. The upregulation of HO‐1 is attributed to heme derived from haemoglobin in RBCEVs. Heme is released from internalized RBCEVs in late endosomes and lysosomes via the heme transporter, HRG1. Consequently, RBCEVs exhibit the ability to attenuate foam cell formation from oxidized low‐density lipoproteins (oxLDL)‐treated macrophages in vitro and reduce atherosclerotic lesions in ApoE knockout mice on a high‐fat diet. In summary, our study reveals the uptake mechanism of RBCEVs and their delivery of heme to macrophages, suggesting the potential application of RBCEVs in the treatment of atherosclerosis. [ABSTRACT FROM AUTHOR]

Background: L-type CaV1.2 channels play crucial roles in the regulation of blood pressure. Galectin-1 (Gal-1) has been reported to bind to the I-II loop of CaV1.2 channels to reduce their current density. However, the mechanistic understanding for the downregulation of CaV1.2 channels by Gal-1 and whether Gal-1 plays a direct role in blood pressure regulation remain unclear.Methods: In vitro experiments involving coimmunoprecipitation, Western blot, patch-clamp recordings, immunohistochemistry, and pressure myography were used to evaluate the molecular mechanisms by which Gal-1 downregulates CaV1.2 channel in transfected, human embryonic kidney 293 cells, smooth muscle cells, arteries from Lgasl1-/- mice, rat, and human patients. In vivo experiments involving the delivery of Tat-e9c peptide and AAV5-Gal-1 into rats were performed to investigate the effect of targeting CaV1.2-Gal-1 interaction on blood pressure monitored by tail-cuff or telemetry methods.Results: Our study reveals that Gal-1 is a key regulator for proteasomal degradation of CaV1.2 channels. Gal-1 competed allosterically with the CaVβ subunit for binding to the I-II loop of the CaV1.2 channel. This competitive disruption of CaVβ binding led to CaV1.2 degradation by exposing the channels to polyubiquitination. It is notable that we demonstrated that the inverse relationship of reduced Gal-1 and increased CaV1.2 protein levels in arteries was associated with hypertension in hypertensive rats and patients, and Gal-1 deficiency induces higher blood pressure in mice because of the upregulated CaV1.2 protein level in arteries. To directly regulate blood pressure by targeting the CaV1.2-Gal-1 interaction, we administered Tat-e9c, a peptide that competed for binding of Gal-1 by a miniosmotic pump, and this specific disruption of CaV1.2-Gal-1 coupling increased smooth muscle CaV1.2 currents, induced larger arterial contraction, and caused hypertension in rats. In contrasting experiments, overexpression of Gal-1 in smooth muscle by a single bolus of AAV5-Gal-1 significantly reduced blood pressure in spontaneously hypertensive rats.Conclusions: We have defined molecularly that Gal-1 promotes CaV1.2 degradation by replacing CaVβ and thereby exposing specific lysines for polyubiquitination and by masking I-II loop endoplasmic reticulum export signals. This mechanistic understanding provided the basis for targeting CaV1.2-Gal-1 interaction to demonstrate clearly the modulatory role that Gal-1 plays in regulating blood pressure, and offering a potential approach for therapeutic management of hypertension. [ABSTRACT FROM AUTHOR]

Low socioeconomic status (SES) is an important prognosticator amongst patients with acute coronary syndrome (ACS). This paper analysed the effects of SES on ACS outcomes. Medline and Embase were searched for articles reporting outcomes of ACS patients stratified by SES using a multidimensional index, comprising at least 2 of the following components: Income, Education and Employment. A comparative meta-analysis was conducted using random-effects models to estimate the risk ratio of all-cause mortality in low SES vs high SES populations, stratified according to geographical region, study year, follow-up duration and SES index. A total of 29 studies comprising of 301,340 individuals were included, of whom 43.7% were classified as low SES. While patients of both SES groups had similar cardiovascular risk profiles, ACS patients of low SES had significantly higher risk of all-cause mortality (adjusted HR:1.19, 95%CI: 1.10–1.1.29, p < 0.001) compared to patients of high SES, with higher 1-year mortality (RR:1.08, 95%CI:1.03–1.13, p = 0.0057) but not 30-day mortality (RR:1.07, 95%CI:0.98–1.16, p = 0.1003). Despite having similar rates of ST-elevation myocardial infarction and non-ST-elevation ACS, individuals with low SES had lower rates of coronary revascularisation (RR:0.95, 95%CI:0.91–0.99, p = 0.0115) and had higher cerebrovascular accident risk (RR:1.25, 95%CI:1.01–1.55, p = 0.0469). Excess mortality risk was independent of region (p = 0.2636), study year (p = 0.7271) and duration of follow-up (p = 0.0604) but was dependent on the SES index used (p < 0.0001). Low SES is associated with increased mortality post-ACS, with suboptimal coronary revascularisation rates compared to those of high SES. Concerted efforts are needed to address the global ACS-related socioeconomic inequity. The current study was registered with PROSPERO, ID: CRD42022347987. [Display omitted] • Socioeconomic deprivation affects Acute Coronary Syndrome (ACS) outcomes and quality of care received. • The low SES group had significantly higher risk of all-cause mortality and lower rates of coronary revascularisation. • There was a disproportionately higher number of females and individuals with diabetes in the low SES group. • There are statistically significant differences in the results drawn based on the socioeconomic index used. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs), which are miniaturised carriers loaded with functional proteins, lipids, and nucleic acid material, are naturally secreted by cells and show intrinsic pharmacological effects in several conditions. As such, they have the potential to be used for the treatment of various human diseases. However, the low isolation yield and laborious purification process are obstacles to their translation for clinical use. To overcome this problem, our lab developed cell-derived nanovesicles (CDNs), which are EV mimetics produced by shearing cells through membrane-fitted spin cups. To evaluate the similarities between EVs and CDNs, we compare the physical properties and biochemical composition of monocytic U937 EVs and U937 CDNs. Besides having similar hydrodynamic diameters, the produced CDNs had proteomic, lipidomic, and miRNA profiles with key communalities compared to those of natural EVs. Further characterisation was conducted to examine if CDNs could exhibit similar pharmacological activities and immunogenicity when administered in vivo. Consistently, CDNs and EVs modulated inflammation and displayed antioxidant activities. EVs and CDNs both did not exert immunogenicity when administered in vivo. Overall, CDNs could serve as a scalable and efficient alternative to EVs for further translation into clinical use. [ABSTRACT FROM AUTHOR]

The subset of plasma extracellular vesicles (EVs) that coprecipitate with low-density lipoprotein (LDL-EVs) carry coagulation and fibrinolysis pathway proteins as cargo. We investigated the association between LDL-EV hemostatic/fibrinolysis protein ratios and post-acute myocardial infarction (post-AMI) left ventricular (LV) remodeling which precedes heart failure. Protein concentrations of von Willebrand factor (VWF), SerpinC1 and plasminogen were determined in LDL-EVs extracted from plasma samples obtained at baseline (within 72 h post-AMI), 1 month and 6 months post-AMI from 198 patients. Patients were categorized as exhibiting adverse (n = 98) or reverse (n = 100) LV remodeling based on changes in LV end-systolic volume (increased or decreased ≥15) over a 6-month period. Multiple level longitudinal data analysis with structural equation (ML-SEM) model was used to assess predictive value for LV remodeling independent of baseline differences. At baseline, protein levels of VWF, SerpinC1 and plasminogen in LDL-EVs did not differ between patients with adverse versus reverse LV remodeling. At 1 month post-AMI, protein levels of VWF and SerpinC1 decreased whilst plasminogen increased in patients with adverse LV remodeling. In contrast, VWF and plasminogen decreased whilst SerpinC1 remained unchanged in patients with reverse LV remodeling. Overall, compared with patients with adverse LV remodeling, higher levels of SerpinC1 and VWF but lower levels of plasminogen resulted in higher ratios of VWF:Plasminogen and SerpinC1:Plasminogen at both 1 month and 6 months post-AMI in patients with reverse LV remodeling. More importantly, ratios VWF:Plasminogen (AUC = 0.674) and SerpinC1:Plasminogen (AUC = 0.712) displayed markedly better prognostic power than NT-proBNP (AUC = 0.384), troponin-I (AUC = 0.467) or troponin-T (AUC = 0.389) (p < 0.001) to predict reverse LV remodeling post-AMI. Temporal changes in the ratios of coagulation to fibrinolysis pathway proteins in LDL-EVs outperform current standard plasma biomarkers in predicting post-AMI reverse LV remodeling. Our findings may provide clinical cues to uncover the cellular mechanisms underpinning post-AMI reverse LV remodeling. [ABSTRACT FROM AUTHOR]

In drug delivery, the development of nanovesicles that combine both synthetic and cellular components provides added biocompatibility and targeting specificity in comparison to conventional synthetic carriers such as liposomes. Produced through the fusion of U937 monocytes' membranes and synthetic lipids, our nano-cell vesicle technology systems (nCVTs) showed promising results as targeted cancer treatment. However, no investigation has been conducted yet on the immunogenic profile and the uptake mechanisms of nCVTs. Hence, this study was aimed at exploring the potential cytotoxicity and immune cells' activation by nCVTs, as well as the routes through which cells internalize these biohybrid systems. The endocytic pathways were selectively inhibited to establish if the presence of cellular components in nCVTs affected the internalization route in comparison to both liposomes (made up of synthetic lipids only) and nano-cellular membranes (made up of biological material only). As a result, nCVTs showed an 8-to-40-fold higher cellular internalization than liposomes within the first hour, mainly through receptor-mediated processes (i.e., clathrin- and caveolae-mediated endocytosis), and low immunostimulatory potential (as indicated by the level of IL-1α, IL-6, and TNF-α cytokines) both in vitro and in vivo. These data confirmed that nCVTs preserved surface cues from their parent U937 cells and can be rationally engineered to incorporate ligands that enhance the selective uptake and delivery toward target cells and tissues. [ABSTRACT FROM AUTHOR]

The therapeutic potential of antigen-specific regulatory T cells (Treg) has been extensively explored, leading to the development of several tolerogenic vaccines. Dexamethasone-antigen conjugates represent a prominent class of tolerogenic vaccines that enable coordinated delivery of antigen and dexamethasone to target immune cells. The importance of nonspecific albumin association towards the biodistribution of antigen-adjuvant conjugates has gained increasing attention, by which hydrophobic and electrostatic interactions govern the association capacity. Using an ensemble of computational and experimental techniques, we evaluate the impact of charged residues adjacent to the drug conjugation site in dexamethasone-antigen conjugates (Dex-K/E4-OVA323, K: lysine, E: glutamate) towards their albumin association capacity and induction of antigen-specific Treg. We find that Dex-K4-OVA323 possesses a higher albumin association capacity than Dex-E4-OVA323, leading to enhanced liver distribution and antigen-presenting cell uptake. Furthermore, using an OVA323-specific adoptive-transfer mouse model, we show that Dex-K4-OVA323 selectively upregulated OVA323-specific Treg cells, whereas Dex-E4-OVA323 exerted no significant effect on Treg cells. Our findings serve as a guide to optimize the functionality of dexamethasone-antigen conjugate amid switching vaccine epitope sequences. Moreover, our study demonstrates that moderating the residues adjacent to the conjugation sites can serve as an engineering approach for future peptide-drug conjugate development. [Display omitted] [ABSTRACT FROM AUTHOR]

The most common chronic liver illness, non-alcoholic fatty liver disease (NAFLD), refers to a range of abnormalities of the liver with varying degrees of steatosis. When the clinical symptoms including liver damage, inflammation, and fibrosis, are added to the initial steatosis, NAFLD becomes non-alcoholic steatohepatitis (NASH), the problematic and severe stage. The diagnosis of NASH at the right time could therefore effectively prevent deterioration of the disease. Considering that platelets (PLTs) could migrate to the sites of inflamed liver sinusoids with oxidative stress during the development of NASH, we purified the PLTs from fresh blood and engineered their surface with hydrogen peroxide (H 2 O 2) responsive fluorescent probe (5-DP) through lipid fusion. The engineered PLT-DPs were recruited and trapped in the inflammation foci of the liver with NASH through interaction with the extracellular matrix, including hyaluronan and Kupffer cells. Additionally, the fluorescence of 5-DP on the surface of PLT-DP was significantly enhanced upon reacting with the elevated level of H 2 O 2 in the NASH liver. Thus, PLT-DP has great promise for NASH fluorescence imaging with high selectivity and sensitivity. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are a class of cell-derived lipid-bilayer membrane vesicles secreted by almost all mammalian cells and involved in intercellular communication by shuttling various biological cargoes. Over the last decade, EVs – namely exosomes and microvesicles – have been extensively explored as next-generation nanoscale drug delivery systems (DDSs). This is in large due to their endogenous origin, which enables EVs to circumvent some of the limitations associated with existing cancer therapy approaches (i.e. by preventing recognition by the immune system and improving selectivity towards tumor tissue). However, successful translation of these cell-derived vesicles into clinical applications has been hindered by several factors, among which the loading of exogenous therapeutic molecules still represents a great challenge. In order to address this issue and to further advance these biologically-derived systems as drug carriers, EV-biohybrid nano-DDSs, obtained through the fusion of EVs with conventional synthetic nano-DDSs, have recently been proposed as a valuable alternative as DDSs. Building on the idea of "combining the best of both worlds", a combination of these two unique entities aims to harness the beneficial properties associated with both EVs and conventional nano-DDSs, while overcoming the flaws of the individual components. These biohybrid systems also provide a unique opportunity for exploitation of new synergisms, often leading to improved therapeutic outcomes, thus paving the way for advancements in cancer therapy. This review aims to describe the recent developments of EV-biohybrid nano-DDSs in cancer therapy, to highlight the most promising results and breakthroughs, as well as to provide a glimpse on the possible intrinsic targeting mechanisms of EVs that can be bequeathed to their hybrid systems. Finally, we also provide some insights in the future perspectives of EV-hybrid DDSs. [ABSTRACT FROM AUTHOR]

Secretome derived from human amniotic fluid stem cells (AFSC-S) is rich in soluble bioactive factors (SBF) and offers untapped therapeutic potential for regenerative medicine while avoiding putative cell-related complications. Characterization and optimal generation of AFSC-S remains challenging. We hypothesized that modulation of oxygen conditions during AFSC-S generation enriches SBF and confers enhanced regenerative and cardioprotective effects on cardiovascular cells. We collected secretome at 6-hourly intervals up to 30 h following incubation of AFSC in normoxic (21%O2, nAFSC-S) and hypoxic (1%O2, hAFSC-S) conditions. Proliferation of human adult cardiomyocytes (hCM) and umbilical cord endothelial cells (HUVEC) incubated with nAFSC-S or hAFSC-S were examined following culture in normoxia or hypoxia. Lower AFSC counts and richer protein content in AFSC-S were observed in hypoxia. Characterization of AFSC-S by multiplex immunoassay showed higher concentrations of pro-angiogenic and anti-inflammatory SBF. hCM demonstrated highest proliferation with 30h-hAFSC-S in hypoxic culture. The cardioprotective potential of concentrated 30h-hAFSC-S treatment was demonstrated in a myocardial ischemia–reperfusion injury mouse model by infarct size and cell apoptosis reduction and cell proliferation increase when compared to saline treatment controls. Thus, we project that hypoxic-generated AFSC-S, with higher pro-angiogenic and anti-inflammatory SBF, can be harnessed and refined for tailored regenerative applications in ischemic cardiovascular disease. [ABSTRACT FROM AUTHOR]

Over the years, the performance of the liposomal formulations of temoporfin, Foslip® and Fospeg®, was investigated in a broad array of cell-based assays and preclinical animal models. So far, little attention has been paid to the influence of drug release and liposomal stability on the plasma concentration–time profile. The drug release is a key attribute which impacts product quality and the in vivo efficacy of nanocarrier formulations. In the present approach, the in vitro drug release and the drug-protein transfer of Foslip® and Fospeg® was determined using the dispersion releaser technology. To analyze the stability of both formulations in physiological fluids, nanoparticle tracking analysis was applied. A comparable drug release behavior and a high physical stability with a vesicle size of approximately 92 ± 2 nm for Foslip® and at 111 ± 5 nm for Fospeg® were measured. The development of a novel hybrid in silico model resulted in an optimal representation of the in vivo data. Based on the information available for previous formulations, the model enabled a prediction of the performance of Foslip® in humans. To verify the simulations, plasma concentration–time profiles of a phase I clinical trial were used. An absolute average fold error of 1.4 was achieved. Moreover, a deconvolution of the pharmacokinetic profile into different fractions relevant for the in vivo efficacy and safety was achieved. While the total plasma concentration reached a c max of 2298 ng/mL after 0.72 h, the monomolecular drug accounted for a small fraction of the photosensitizer with a c max of 321 ng/mL only. [ABSTRACT FROM AUTHOR]

Aims The Toll-like receptor 7 (TLR7) is an intracellular innate immune receptor activated by nucleic acids shed from dying cells leading to activation of the innate immune system. Since innate immune system activation is involved in the response to myocardial infarction (MI), this study aims to identify if TLR7 is involved in post-MI ischaemic injury and adverse remodelling after MI. Methods and results TLR7 involvement in MI was investigated in human tissue from patients with ischaemic heart failure, as well as in a mouse model of permanent left anterior descending artery occlusion in C57BL/6J wild type and TLR7 deficient (TLR7−/−) mice. TLR7 expression was up-regulated in human and mouse ischaemic myocardium after MI. Compared to wild type mice, TLR7−/− mice had less acute cardiac rupture associated with blunted activation of matrix metalloproteinase 2, increased expression of tissue inhibitor of metalloproteinase 1, recruitment of more myofibroblasts, and the formation of a myocardial scar with higher collagen fibre density. Furthermore, inflammatory cell influx and inflammatory cytokine expression post-MI were reduced in the TLR7−/− heart. During a 28-day follow-up after MI, TLR7 deficiency resulted in less chronic adverse left ventricular remodelling and better cardiac function. Bone marrow (BM) transplantation experiments showed that TLR7 deficiency in BM-derived cells preserved cardiac function after MI. Conclusions In acute MI, TLR7 mediates the response to acute cardiac injury and chronic remodelling probably via modulation of post-MI scar formation and BM-derived inflammatory infiltration of the myocardium. [ABSTRACT FROM AUTHOR]

Abstract Background Statins are thought to have pleiotropic properties, including anticoagulant effects, in addition to reducing lipoprotein (LDL) levels. Plasma extracellular vesicles (EVs) are small bilayer membrane vesicles involved in various biological processes including coagulation. Since subsets of EVs in the LDL plasma fraction (LDL-EVs) correlate with thrombin activity, we hypothesized that changes in LDL-EVs after statin therapy may differ from that of serum levels of coagulation proteins, providing insight into the effects of statins on coagulation. Methods The study was conducted in 666 subjects with available serum from the METEOR trial, a trial of the effect of rosuvastatin versus placebo in patients with subclinical atherosclerosis. Changes in protein levels of von Willebrand Factor (VWF), SerpinC1 and plasminogen were measured in serum and in LDL-EVs, and were compared between the rosuvastatin and placebo groups. Results LDL-EV levels of plasminogen and VWF increased with rosuvastatin treatment compared to placebo (mean change of 126 ± 8 versus 17 ± 12 μg/mL for plasminogen (p < 0.001) and 310 ± 60 versus 64 ± 55 μg/mL for VWF (p = 0.015)). There was no difference between groups for change in LDL-EV-SerpinC1. In contrast, serum plasminogen levels increased to a lesser extent with rosuvastatin compared to placebo (23 ± 29 versus 67 ± 17 μg/mL, p = 0.024) and serum VWF levels showed no significant difference between both groups. Conclusions Rosuvastatin increases LDL-EV coagulation proteins plasminogen and VWF in patients with subclinical atherosclerosis, an effect that is different from the effect of rosuvastatin on the same proteins in serum. This identifies LDL-EVs as a newly detected possible intermediate between statin therapy and coagulation. Highlights • LDL associated extracellular vesicles (LDL-EVs) are involved coagulation processes. • This study investigates the effect of statins on coagulation proteins in LDL-EVs. • Rosuvastatin increases plasminogen and von Willebrand factor in LDL-EVs. • LDL-EVs are identified as a possible intermediate between statins and coagulation. [ABSTRACT FROM AUTHOR]

Inflammation is a known mediator of adverse ventricular remodeling after myocardial infarction (MI) that may lead to reduction of ejection fraction and subsequent heart failure. Berberine is a isoquinoline quarternary alkaloid from plants that has been associated with anti-inflammatory, anti-oxidative, and cardioprotective properties. Its poor solubility in aqueous buffers and its short half-life in the circulation upon injection, however, have been hampering the extensive usage of this natural product. We hypothesized that encapsulation of berberine into long circulating liposomes could improve its therapeutic availability and efficacy by protecting cardiac function against MI in vivo. Berberine-loaded liposomes were prepared by ethanol injection and characterized. They contained 0.3 mg/mL of the drug and were 0.11 μm in diameter. Subsequently they were tested for IL-6 secretion inhibition in RAW 264.7 macrophages and for cardiac function protection against adverse remodeling after MI in C57BL/6J mice. In vitro , free berberine significantly inhibited IL-6 secretion (IC 50 = 10.4 μM), whereas encapsulated berberine did not as it was not released from the formulation in the time frame of the in vitro study. In vivo , berberine-loaded liposomes significantly preserved the cardiac ejection fraction at day 28 after MI by 64% as compared to control liposomes and free berberine. In conclusion, liposomal encapsulation enhanced the solubility of berberine in buffer and preserves ejection fraction after MI. This shows that delivery of berberine-loaded liposomes significantly improves its therapeutic availability and identifies berberine-loaded liposomes as potential treatment of adverse remodeling after MI. [ABSTRACT FROM AUTHOR]

Pulsing electromagnetic fields (PEMFs) have been shown to promote in vitro and in vivo myogeneses via mitohormetic survival adaptations of which secretome activation is a key component. A single 10-min exposure of donor myoblast cultures to 1.5 mT amplitude PEMFs produced a conditioned media (pCM) capable of enhancing the myogenesis of recipient cultures to a similar degree as direct magnetic exposure. Downwardly-directed magnetic fields produced greater secretome responses than upwardly-directed fields in adherent and fluid-suspended myoblasts. The suspension paradigm allowed for the rapid concentrating of secreted factors, particularly of extracellular vesicles. The brief conditioning of basal media from magnetically-stimulated myoblasts was capable of conferring myoblast survival to a greater degree than basal media supplemented with fetal bovine serum (5%). Downward-directed magnetic fields, applied directly to cells or in the form of pCM, upregulated the protein expression of TRPC channels, markers for cell cycle progression and myogenesis. Direct magnetic exposure produced mild oxidative stress, whereas pCM provision did not, providing a survival advantage on recipient cells. Streptomycin, a TRP channel antagonist, precluded the production of a myogenic pCM. We present a methodology employing a brief and non-invasive PEMF-exposure paradigm to effectively stimulate secretome production and release for commercial or clinical exploitation. [ABSTRACT FROM AUTHOR]

Background: SerpinF2, SerpinG1, CystatinC and CD14 are involved in inflammatory processes and plasma extracellular vesicle (EV) -levels of these proteins have been reported to be associated with systemic vascular events. Evidence is accumulating that inflammatory processes may play a pivotal role both in systemic vascular events and in heart failure. Therefore, we studied the association between plasma extracellular vesicle SerpinF2-, SerpinG1-, CystatinC and CD14-levels and the occurrence of acute heart failure in patients. Methods and Result: Extracellular vesicle protein levels of SerpinG1, SerpinF2, CystatinC and CD14 were measured in an observational study of 404 subjects presenting with dysponea at the emergency department (4B-cohort). Plasma extracellular vesicles were precipitated in a total extracellular vesicles (TEX)-fraction and in separate LDL- and HDL-subfractions. Extracellular vesicle protein levels were measured with a quantitative immune assay in all 3 precipitates. Out of 404 subjects, 141 (35%) were diagnosed with acutely decompensated heart failure. After correction for confounders (including comorbidities and medications), levels of CD14 in the HDL-fraction (OR 1.53, p = 0.01), SerpinF2 in the TEX-and LDL-fraction (ORs respectively 0.71 and 0.65, p<0.05) and SerpinG1 in the TEX-fraction (OR 1.55, p = 0.004) were statistically significantly related to heart failure. Furthermore, extracellular vesicle CD14- and SerpinF2-levels were significantly higher in heart failure patients with preserved ejection fraction than in those with reduced ejection fraction. Conclusion: Extracellular vesicle levels of CD14, SerpinG1 and SerpinF2 are associated with the occurrence of heart failure in subjects suspected for acute heart failure, suggesting common underlying pathophysiological mechanisms for heart failure and vascular events. [ABSTRACT FROM AUTHOR]

[Display omitted] For many years, nanomedicine is pushing the boundaries of drug delivery. When applying these novel therapeutics, safety considerations are not only a key concern when entering clinical trials but also an important decision point in product development. Standing at the crossroads, nanomedicine may be able to escape the niche markets and achieve wider acceptance by the pharmaceutical industry. While there is a new generation of drug delivery systems, the extracellular vesicles, standing on the starting line, unresolved issues and new challenges emerge from their translation from bench to bedside. Some key features of injectable nanomedicines contribute to the predictability of the pharmacological and toxicological effects. So far, only a few of the physicochemical attributes of nanomedicines can be justified by a direct mathematical relationship between the in vitro and the in vivo responses. To further develop extracellular vesicles as drug carriers, we have to learn from more than 40 years of clinical experience in liposomal delivery and pass on this knowledge to the next generation. Our quick guide discusses relationships between physicochemical characteristics and the in vivo response, commonly referred to as in vitro-in vivo correlation. Further, we highlight the key role of computational methods, lay open current knowledge gaps, and question the established design strategies. Has the recent progress improved the predictability of targeted delivery or do we need another change in perspective? [ABSTRACT FROM AUTHOR]

[Display omitted] • Pros and cons and recent trends of EVs for drug delivery in animal disease models. • Quantitative analysis of the key parameters of EVs as drug delivery systems. • EVs have unique niches for RNA delivery and vaccine development. • Preclinical data of EVs as drug delivery systems to interpret with caution. • Lack of regulatory guidelines for clinical research of EVs as drug delivery systems. During the past decades, extracellular vesicles (EVs) have emerged as an attractive drug delivery system. Here, we assess their pre-clinical applications, in the form of a systematic review. For each study published in the past decade, disease models, animal species, EV donor cell types, active pharmaceutical ingredients (APIs), EV surface modifications, API loading methods, EV size and charge, estimation of EV purity, presence of biodistribution studies and administration routes were quantitatively analyzed in a defined and reproducible way. We have interpreted the trends we observe over the past decade, to define the niches where to apply EVs for drug delivery in the future and to provide a basis for regulatory guidelines. [ABSTRACT FROM AUTHOR]

[Display omitted] Fibrosis is a common denominator in many pathologies and crucially affects disease progression, drug delivery efficiency and therapy outcome. We here summarize therapeutic and diagnostic strategies for fibrosis targeting in atherosclerosis and cardiac disease, cancer, diabetes, liver diseases and viral infections. We address various anti-fibrotic targets, ranging from cells and genes to metabolites and proteins, primarily focusing on fibrosis-promoting features that are conserved among the different diseases. We discuss how anti-fibrotic therapies have progressed over the years, and how nanomedicine formulations can potentiate anti-fibrotic treatment efficacy. From a diagnostic point of view, we discuss how medical imaging can be employed to facilitate the diagnosis, staging and treatment monitoring of fibrotic disorders. Altogether, this comprehensive overview serves as a basis for developing individualized and improved treatment strategies for patients suffering from fibrosis-associated pathologies. [ABSTRACT FROM AUTHOR]

Recently, bioinspired cell-derived nanovesicles (CDNs) have gained much interest in the field of nanomedicine due to the preservation of biomolecular structure characteristics derived from their parent cells, which impart CDNs with unique properties in terms of binding and uptake by target cells and intrinsic biological activities. Although the production of CDNs can be easily and reproducibly achieved with any kind of cell culture, application of CDNs for therapeutic purposes has been greatly hampered by their physical and chemical instability during long-term storage in aqueous dispersion. In the present study, we conceived a lyophilization approach that would preserve critical characteristics regarding stability (vesicles' size and protein content), structural integrity, and biological activity of CDNs for enabling long-term storage in freeze-dried form. Compared to the lyoprotectant sucrose, trehalose-lyoprotected CDNs showed significantly higher glass transition temperature and lower residual moisture content. As assessed by ATR-FTIR and far-UV circular dichroism, lyophilization in the presence of the lyoprotectant effectively maintained the secondary structure of cellular proteins. After reconstitution, lyoprotected CDNs were efficiently associated with HeLa cells, CT26 cells, and bone marrow-derived macrophages at a rate comparable to the freshly prepared CDNs. In vivo, both lyoprotected and freshly prepared CDNs, for the first time ever reported, targeted the injured heart, and exerted intrinsic cardioprotective effects within 24 h, attributable to the antioxidant capacity of CDNs in a myocardial ischemia/reperfusion injury animal model. Taken together, these results pave the way for further development of CDNs as cell-based therapeutics stabilized by lyophilization that enabled long-term storage while preserving their activity. [ABSTRACT FROM AUTHOR]

Cardiovascular diseases (CVD) represent the leading cause of morbidity and mortality globally. The emerging role of extracellular vesicles (EVs) in intercellular communication has stimulated renewed interest in exploring the potential application of EVs as tools for diagnosis, prognosis, and therapy in CVD. The ubiquitous nature of EVs in biological fluids presents a technological advantage compared to current diagnostic tools by virtue of their notable stability. EV contents, such as proteins and microRNAs, represent specific signatures of cellular activation or injury. This feature positions EVs as an alternative source of biomarkers. Furthermore, their intrinsic activity and immunomodulatory properties offer EVs unique opportunities to act as therapeutic agents per se or to serve as drug delivery carriers by acting as miniaturized vehicles incorporating bioactive molecules. In this article, we aim to review the recent advances and applications of EV-based biomarkers and therapeutics. In addition, the potential of EVs as a drug delivery and theranostic platform for CVD will also be discussed. [ABSTRACT FROM AUTHOR]