Expression and Function of Connexins in Extracellular Vesicles.

Extracellular vesicles (EVs) are recognized as major vehicles for exchange of information across distant cells and tissues, which have been extensively explored for diagnosis and therapeutic purposes. The presence of multiple connexin (Cx) proteins has been described in EVs, where they might facilitate EV-cell communication. However, quantitative changes in Cx levels and functional assessment of Cx channels have only been established for Cx43. In present work, we provide a detailed description of the protocols we have optimized to assess the expression and permeability of Cx43 channels in EVs derived from cultured cells and human peripheral blood. Particularly, we include some modifications to improve quantitative analysis of EV-Cx43 by enzyme-linked immunosorbent assay (ELISA) and assessment of channel functionality by sucrose-density gradient ultracentrifugation, which can be easily adapted to other Cx family members, leveraging the development of diagnostic and therapeutic applications based on Cx-containing EVs.

Effect of Blueberry Supplementation on a Diet-Induced Rat Model of Prediabetes-Focus on Hepatic Lipid Deposition, Endoplasmic Stress Response and Autophagy.

Blueberries, red fruits enriched in polyphenols and fibers, are envisaged as a promising nutraceutical intervention in a plethora of metabolic diseases. Prediabetes, an intermediate state between normal glucose tolerance and type 2 diabetes, fuels the development of complications, including hepatic steatosis. In previous work, we have demonstrated that blueberry juice (BJ) supplementation benefits glycemic control and lipid profile, which was accompanied by an amelioration of hepatic mitochondrial bioenergetics. The purpose of this study is to clarify the impact of long-term BJ nutraceutical intervention on cellular mechanisms that govern hepatic lipid homeostasis, namely autophagy and endoplasmic reticulum (ER) stress, in a rat model of prediabetes. Two groups of male Wistar rats, 8-weeks old, were fed a prediabetes-inducing high-fat diet (HFD) and one group was fed a control diet (CD). From the timepoint where the prediabetic phenotype was achieved (week 16) until the end of the study (week 24), one of the HFD-fed groups was daily orally supplemented with 25 g/kg body weight (BW) of BJ (HFD + BJ). BW, caloric intake, glucose tolerance and insulin sensitivity were monitored throughout the study. The serum and hepatic lipid contents were quantified. Liver and interscapular brown and epidydimal white adipose tissue depots (iBAT and eWAT) were collected for histological analysis and to assess thermogenesis, ER stress and autophagy markers. The gut microbiota composition and the short-chain fatty acids (SCFAs) content were determined in colon fecal samples. BJ supplementation positively impacted glycemic control but was unable to prevent obesity and adiposity. BJ-treated animals presented a reduction in fecal SCFAs, increased markers of arrested iBAT thermogenesis and energy expenditure, together with an aggravation of HFD-induced lipotoxicity and hepatic steatosis, which were accompanied by the inhibition of autophagy and ER stress responses in the liver. In conclusion, despite the improvement of glucose tolerance, BJ supplementation promoted a major impact on lipid management mechanisms at liver and AT levels in prediabetic animals, which might affect disease course.

Unveiling the role of osteosarcoma-derived secretome in premetastatic lung remodelling.

BACKGROUND: Lung metastasis is the most adverse clinical factor and remains the leading cause of osteosarcoma-related death. Deciphering the mechanisms driving metastatic spread is crucial for finding open therapeutic windows for successful organ-specific interventions that may halt or prevent lung metastasis. METHODS: We employed a mouse premetastatic lung-based multi-omics integrative approach combined with clinical features to uncover the specific changes that precede lung metastasis formation and identify novel molecular targets and biomarker of clinical utility that enable the design of novel therapeutic strategies. RESULTS: We found that osteosarcoma-bearing mice or those preconditioned with the osteosarcoma cell secretome harbour profound lung structural alterations with airway damage, inflammation, neutrophil infiltration, and extracellular matrix remodelling with increased deposition of fibronectin and collagens by resident stromal activated fibroblasts, favouring the adhesion of disseminated tumour cells. Systemic-induced microenvironmental changes, supported by transcriptomic and histological data, promoted and accelerated lung metastasis formation. Comparative proteome profiling of the cell secretome and mouse plasma identified a large number of proteins involved in extracellular-matrix organization, cell-matrix adhesion, neutrophil degranulation, and cytokine-mediated signalling, consistent with the observed lung microenvironmental changes. Moreover, we identified EFEMP1, an extracellular matrix glycoprotein exclusively secreted by metastatic cells, in the plasma of mice bearing a primary tumour and in biopsy specimens from osteosarcoma patients with poorer overall survival. Depletion of EFEMP1 from the secretome prevents the formation of lung metastasis. CONCLUSIONS: Integration of our data uncovers neutrophil infiltration and the functional contribution of stromal-activated fibroblasts in ECM remodelling for tumour cell attachment as early pro-metastatic events, which may hold therapeutic potential in preventing or slowing the metastatic spread. Moreover, we identified EFEMP1, a secreted glycoprotein, as a metastatic driver and a potential candidate prognostic biomarker for lung metastasis in osteosarcoma patients. Osteosarcoma-derived secreted factors systemically reprogrammed the lung microenvironment and fostered a growth-permissive niche for incoming disseminated cells to survive and outgrow into overt metastasis. Daily administration of osteosarcoma cell secretome mimics the systemic release of tumour-secreted factors of a growing tumour in mice during PMN formation; Transcriptomic and histological analysis of premetastatic lungs revealed inflammatory-induced stromal fibroblast activation, neutrophil infiltration, and ECM remodelling as early onset pro-metastatic events; Proteome profiling identified EFEMP1, an extracellular secreted glycoprotein, as a potential predictive biomarker for lung metastasis and poor prognosis in osteosarcoma patients. Osteosarcoma patients with EFEMP1 expressing biopsies have a poorer overall survival.

Cx43 can form functional channels at the nuclear envelope and modulate gene expression in cardiac cells.

Classically associated with gap junction-mediated intercellular communication, connexin43 (Cx43) is increasingly recognized to possess non-canonical biological functions, including gene expression regulation. However, the mechanisms governing the localization and role played by Cx43 in the nucleus, namely in transcription modulation, remain unknown. Using comprehensive and complementary approaches encompassing biochemical assays, super-resolution and immunogold transmission electron microscopy, we demonstrate that Cx43 localizes to the nuclear envelope of different cell types and in cardiac tissue. We show that translocation of Cx43 to the nucleus relies on Importin-ß, and that Cx43 significantly impacts the cellular transcriptome, likely by interacting with transcriptional regulators. In vitro patch-clamp recordings from HEK293 and adult primary cardiomyocytes demonstrate that Cx43 forms active channels at the nuclear envelope, providing evidence that Cx43 can participate in nucleocytoplasmic shuttling of small molecules. The accumulation of nuclear Cx43 during myogenic differentiation of cardiomyoblasts is suggested to modulate expression of genes implicated in this process. Altogether, our study provides new evidence for further defining the biological roles of nuclear Cx43, namely in cardiac pathophysiology.

Editorial of the Special Issue: Cellular Mechanisms of Cardiovascular Disease.

Cardiovascular diseases (CVD) remain the major cause of mortality and disability worldwide, having contributed to 19 [...].

The good, the bad and the ugly: the impact of extracellular vesicles on the cardiovascular system.

Cardiovascular diseases (CVDs), which encompass a myriad of pathological conditions that affect the heart and/or the blood vessels, remain the major cause of morbidity and mortality worldwide. By transferring a wide variety of bioactive molecules, including proteins and microRNAs (miRNAs), extracellular vesicles (EVs) are recognized as key players in long-range communication across the cardiovascular system. It has been demonstrated that these highly heterogeneous nanosized vesicles participate both in the maintenance of homeostasis of the heart and vessels, and contribute to the pathophysiology of CVDs, thus emerging as promising tools for diagnosis, prognosis and treatment of multiple CVDs. In this review, we highlight the beneficial roles of EV-mediated communication in regulating vascular homeostasis, and inter-organ crosstalk as a potential mechanism controlling systemic metabolic fitness. In addition, the impact of EV secretion in disease development is described, particularly focusing on cardiac remodelling following ischaemia, atherogenesis and atrial fibrillation progression. Finally, we discuss the potential of endogenous and bioengineered EVs as therapeutic tools for CVDs, as well as the suitability of assessing the molecular signature of circulating EVs as a non-invasive predictive marker of CVD onset and progression. This rapidly expanding field of research has established the role of EVs as key conveyors of both cardioprotective and detrimental signals, which might be of relevance in uncovering novel therapeutic targets and biomarkers of CVDs.

Osteosarcoma-Derived Exosomes as Potential PET Imaging Nanocarriers for Lung Metastasis.

Lung metastases represent the most adverse clinical factor and rank as the leading cause of osteosarcoma-related death. Nearly 80% of patients present lung micrometastasis at diagnosis not detected with current clinical tools. Herein, an exosome (EX)-based imaging tool is developed for lung micrometastasis by positron emission tomography (PET) using osteosarcoma-derived EXs as natural nanocarriers of the positron-emitter copper-64 (64 Cu). Exosomes are isolated from metastatic osteosarcoma cells and functionalized with the macrocyclic chelator NODAGA for complexation with 64 Cu. Surface functionalization has no effect on the physicochemical properties of EXs, or affinity for donor cells and endows them with favorable pharmacokinetics for in vivo studies. Whole-body PET/magnetic resonance imaging (MRI) images in xenografted models show a specific accumulation of 64 Cu-NODAGA-EXs in metastatic lesions as small as 2-3 mm or in a primary tumor, demonstrating the exquisite tropism of EXs for homotypic donor cells. The targetability for lung metastasis is also observed by optical imaging using indocyanine green (ICG)-labeled EXs and D-luciferin-loaded EXs. These findings show that tumor-derived EXs hold great potential as targeted imaging agents for the noninvasive detection of small lung metastasis by PET. This represents a step forward in the biomedical application of EXs in imaging diagnosis with increased translational potential.

The Footprint of Exosomes in the Radiation-Induced Bystander Effects.

Radiation therapy is widely used as the primary treatment option for several cancer types. However, radiation therapy is a nonspecific method and associated with significant challenges such as radioresistance and non-targeted effects. The radiation-induced non-targeted effects on nonirradiated cells nearby are known as bystander effects, while effects far from the ionising radiation-exposed cells are known as abscopal effects. These effects are presented as a consequence of intercellular communications. Therefore, a better understanding of the involved intercellular signals may bring promising new strategies for radiation risk assessment and potential targets for developing novel radiotherapy strategies. Recent studies indicate that radiation-derived extracellular vesicles, particularly exosomes, play a vital role in intercellular communications and may result in radioresistance and non-targeted effects. This review describes exosome biology, intercellular interactions, and response to different environmental stressors and diseases, and focuses on their role as functional mediators in inducing radiation-induced bystander effect (RIBE).

Cx43-mediated sorting of miRNAs into extracellular vesicles.

Through the exchange of lipids, proteins, and nucleic acids, extracellular vesicles (EV) allow for cell-cell communication across distant cells and tissues to regulate a wide range of physiological and pathological processes. Although some molecular mediators have been discovered, the mechanisms underlying the selective sorting of miRNAs into EV remain elusive. Previous studies demonstrated that connexin43 (Cx43) forms functional channels at the EV surface, mediating the communication with recipient cells. Here, we show that Cx43 participates in the selective sorting of miRNAs into EV through a process that can also involve RNA-binding proteins. We provide evidence that Cx43 can directly bind to specific miRNAs, namely those containing stable secondary structure elements, including miR-133b. Furthermore, Cx43 facilitates the delivery of EV-miRNAs into recipient cells. Phenotypically, we show that Cx43-mediated EV-miRNAs sorting modulates autophagy. Overall, our study ascribes another biological role to Cx43, that is, the selective incorporation of miRNAs into EV, which potentially modulates multiple biological processes in target cells and may have implications for human health and disease.

Connecting different heart diseases through intercellular communication.

Well-orchestrated intercellular communication networks are pivotal to maintaining cardiac homeostasis and to ensuring adaptative responses and repair after injury. Intracardiac communication is sustained by cell-cell crosstalk, directly via gap junctions (GJ) and tunneling nanotubes (TNT), indirectly through the exchange of soluble factors and extracellular vesicles (EV), and by cell-extracellular matrix (ECM) interactions. GJ-mediated communication between cardiomyocytes and with other cardiac cell types enables electrical impulse propagation, required to sustain synchronized heart beating. In addition, TNT-mediated organelle transfer has been associated with cardioprotection, whilst communication via EV plays diverse pathophysiological roles, being implicated in angiogenesis, inflammation and fibrosis. Connecting various cell populations, the ECM plays important functions not only in maintaining the heart structure, but also acting as a signal transducer for intercellular crosstalk. Although with distinct etiologies and clinical manifestations, intercellular communication derailment has been implicated in several cardiac disorders, including myocardial infarction and hypertrophy, highlighting the importance of a comprehensive and integrated view of complex cell communication networks. In this review, I intend to provide a critical perspective about the main mechanisms contributing to regulate cellular crosstalk in the heart, which may be considered in the development of future therapeutic strategies, using cell-based therapies as a paradigmatic example. This Review has an associated Future Leader to Watch interview with the author.

Cardiac phospholipidome is altered during ischemia and reperfusion in an ex vivo rat model.

Acute myocardial infarction (AMI) is the leading cause of death, morbidity, and health costs worldwide. In AMI, a sudden blockage of blood flow causes myocardial ischemia and cell death. Reperfusion after ischemia has paradoxical effects and may exacerbate the myocardial injury, a process known as ischemic reperfusion injury. In this work we evaluated the lipidome of isolated rat hearts, maintained in controlled perfusion (CT), undergoing global ischemia (ISC) or ischemia followed by reperfusion (IR). 153 polar lipid levels were significantly different between conditions. 48 features had q < 0.001 and included 8 phosphatidylcholines and 4 lysophospholipids, which were lower in ISC compared to CT, and even lower in the IR group, suggesting that IR induces more profound changes than ISC. We observed that the levels of 16 alkyl acyl phospholipids were significantly altered during ISC and IR. Overall, these data indicate that myocardial lipid remodelling and possibly damage occurs to a greater extent during reperfusion. The adaptation of cardiac lipidome during ISC and IR described is consistent with the presence of oxidative damage and may reflect the impact of AMI on the lipidome at the cellular level and provide new insights into the role of lipids in the pathophysiology of acute myocardial ischemia/reperfusion injury.

Cellular crosstalk in cardioprotection: Where and when do reactive oxygen species play a role?

A well-balanced intercellular communication between the different cells within the heart is vital for the maintenance of cardiac homeostasis and function. Despite remarkable advances on disease management and treatment, acute myocardial infarction remains the major cause of morbidity and mortality worldwide. Gold standard reperfusion strategies, namely primary percutaneous coronary intervention, are crucial to preserve heart function. However, reestablishment of blood flow and oxygen levels to the infarcted area are also associated with an accumulation of reactive oxygen species (ROS), leading to oxidative damage and cardiomyocyte death, a phenomenon termed myocardial reperfusion injury. In addition, ROS signaling has been demonstrated to regulate multiple biological pathways, including cell differentiation and intercellular communication. Given the importance of cell-cell crosstalk in the coordinated response after cell injury, in this review, we will discuss the impact of ROS in the different forms of inter- and intracellular communication, as well as the role of gap junctions, tunneling nanotubes and extracellular vesicles in the propagation of oxidative damage in cardiac diseases, particularly in the context of ischemia/reperfusion injury.

RyR2 regulates Cx43 hemichannel intracellular Ca2+-dependent activation in cardiomyocytes.

AIMS: Connexin-based gap junctions are crucial for electrical communication in the heart; they are each composed of two docked hemichannels (HCs), supplied as unpaired channels via the sarcolemma. When open, an unpaired HC forms a large pore, high-conductance and Ca2+-permeable membrane shunt pathway that may disturb cardiomyocyte function. HCs composed of connexin 43 (Cx43), a major cardiac connexin, can be opened by electrical stimulation but only by very positive membrane potentials. Here, we investigated the activation of Cx43 HCs in murine ventricular cardiomyocytes voltage-clamped at -70 mV. METHODS AND RESULTS: Using whole-cell patch-clamp, co-immunoprecipitation, western blot analysis, immunocytochemistry, proximity ligation assays, and protein docking studies, we found that stimulation of ryanodine receptors (RyRs) triggered unitary currents with a single-channel conductance of ∼220 pS, which were strongly reduced by Cx43 knockdown. Recordings under Ca2+-clamp conditions showed that both RyR activation and intracellular Ca2+ elevation were necessary for HC opening. Proximity ligation studies indicated close Cx43-RyR2 apposition (<40 nm), and both proteins co-immunoprecipitated indicating physical interaction. Molecular modelling suggested a strongly conserved RyR-mimicking peptide sequence (RyRHCIp), which inhibited RyR/Ca2+ HC activation but not voltage-triggered activation. The peptide also slowed down action potential repolarization. Interestingly, alterations in the concerned RyR sequence are known to be associated with primary familial hypertrophic cardiomyopathy. CONCLUSION: Our results demonstrate that Cx43 HCs are intimately linked to RyRs, allowing them to open at negative diastolic membrane potential in response to RyR activation.

Intercellular Communication in the Heart: Therapeutic Opportunities for Cardiac Ischemia.

The maintenance of tissue, organ, and organism homeostasis relies on an intricate network of players and mechanisms that assist in the different forms of cell-cell communication. Myocardial infarction, following heart ischemia and reperfusion, is associated with profound changes in key processes of intercellular communication, involving gap junctions, extracellular vesicles, and tunneling nanotubes, some of which have been implicated in communication defects associated with cardiac injury, namely arrhythmogenesis and progression into heart failure. Therefore, intercellular communication players have emerged as attractive powerful therapeutic targets aimed at preserving a fine-tuned crosstalk between the different cardiac cells in order to prevent or repair some of harmful consequences of heart ischemia and reperfusion, re-establishing myocardial function.

Myocardial infarction affects Cx43 content of extracellular vesicles secreted by cardiomyocytes.

Ischemic heart disease has been associated with an impairment on intercellular communication mediated by both gap junctions and extracellular vesicles. We have previously shown that connexin 43 (Cx43), the main ventricular gap junction protein, assembles into channels at the extracellular vesicle surface, mediating the release of vesicle content into target cells. Here, using a comprehensive strategy that included cell-based approaches, animal models and human patients, we demonstrate that myocardial ischemia impairs the secretion of Cx43 into circulating, intracardiac and cardiomyocyte-derived vesicles. In addition, we show that ubiquitin signals Cx43 release in basal conditions but appears to be dispensable during ischemia, suggesting an interplay between ischemia-induced Cx43 degradation and secretion. Overall, this study constitutes a step forward for the characterization of the signals and molecular players underlying vesicle protein sorting, with strong implications on long-range intercellular communication, paving the way towards the development of innovative diagnostic and therapeutic strategies for cardiovascular disorders.